Chimeric Heteromultimer Adhesins

ABSTRACT

Novel chimeric heteromultimer adhesins that bind the ligand of natural heteromultimeric receptors and uses therefor are disclosed. The chimeric molecules of the heteromultimer adhesins comprise an extracellular domain of a heteromultimeric receptor monomer and a multimerization domain for the stable interaction of the chimeric molecules in the adhesin. Specifically disclosed are heteromultimeric adhesins comprising the extracellular domains of ErbB2 and ErbB3 or ErbB2 and ErbB4. The chimeric ErbB heteromultimer adhesins of the present invention are useful as competitive antagonists or agonists of a neuregulin for the treatment of diseases such as various cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a non-provisional application filed under 37 CFR 1.53(b)(1),claiming priority under U.S.C. Section 119(e) to provisional ApplicationSer. No. 60/021,640, filed Jul. 12, 1996.

FIELD OF THE INVENTION

This application relates generally to chimeric heteromultimer adhesinscomprising extracellular binding domains of heteromultimeric receptors,which heteromultimer adhesins bind the ligand of the natural receptor.The invention further relates to antibodies to the heteroadhesins,methods of making the adhesins and methods of using the heteroadhesinsand antibodies.

BACKGROUND OF THE INVENTION

Transduction of signals that regulate cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularsubstrates.

The ErbB family of single-spanning, receptor tyrosine kinases consistsof four members: epidermal growth factor receptor (EGFR), ErbB2(HER2/neu), ErbB3 (HER3) and ErbB4 (HER4). A number of ligands, all ofwhich are different gene products, have been identified that bind andactivate EGFR (reviewed in Groenen et al., 1994). In contrast, a singleneuregulin gene encodes for a large number of protein isoforms thatresult from alternative splicing of mRNA transcripts (reviewed in(Lemke, G. (1996) mol. Cell. Neurosci. 7:247-262). ErbB3 (Carraway, K.L. et al. (1994) J. Biol. Chem. 2:14303-14306) or ErbB4 (Plowman, G. D.et al., (1993) Nature 366:473-475) can serve as receptors for theneuregulins. These receptors and ligands play key roles in normal cellgrowth and differentiation.

Growth factor receptor protein tyrosine kinases of the class I subfamilyinclude the 170 kDa epidermal growth factor receptor (EGFR) encoded bythe erbB1 gene. erbB1 has been causally implicated in human malignancy.In particular, increased expression of this gene has been observed inmore aggressive carcinomas of the breast, bladder, lung and stomach(Modjtahedi, H. and Dean, C. (1994) Int. J. Oncol. 4:277-296).

The second member of the class I subfamily, p185^(neu), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The neu gene (also called erbB2 and HER2)encodes a 185 kDa receptor protein tyrosine kinase. Amplification and/oroverexpression of the human HER2 gene correlates with a poor prognosisin breast and ovarian cancers (Slamon, D. J. et al., Science 235:177-182(1987); and Slamon et al., Science 244:707-712 (1989)). Overexpressionof HER2 has been correlated with other carcinomas including carcinomasof the stomach, endometrium, salivary gland, lung, kidney, colon andbladder. Accordingly, Slamon et al. in U.S. Pat. No. 4,968,603 describeand claim various diagnostic assays for determining HER2 geneamplification or expression in tumor cells. Slamon et al. discoveredthat the presence of multiple gene copies of HER2 oncogene in tumorcells indicates that the disease is more likely to spread beyond theprimary tumor site, and that the disease may therefore require moreaggressive treatment than might otherwise be indicated by otherdiagnostic factors. Slamon et al. conclude that the HER2 geneamplification test, together with the determination of lymph nodestatus, provides greatly improved prognostic utility.

A further related gene, called erbB3 or HER3, has also been described.See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad. Sci. USA86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., Proc.Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discoveredthat markedly elevated levels of erbB3 mRNA were present in certainhuman mammary tumor cell lines indicating that erbB3, like erbB1 anderbB2, may play a role in human malignancies. Also, Kraus et al. (1993)showed that EGF-dependent activation of the ErbB3 catalytic domain of achimeric EGFR/ErbB3 receptor resulted in a proliferative response intransfected NIH-3T3 cells. Furthermore, these researchers demonstratedthat some human mammary tumor cell lines display a significant elevationof steady-state ErbB3 tyrosine phosphorylation further indicating thatthis receptor may play a role in human malignancies. The role of erbB3in cancer has been explored by others. It has been found to beoverexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121(1992)), gastrointestinal (Poller et al., J. Pathol. 168:275-280 (1992),Rajkumer et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int.J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J.Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research1:1413-1420 (1995)).

ErbB3 is unique among the ErbB receptor family in that it possesseslittle or no intrinsic tyrosine kinase activity (Guy et al., Proc. Natl.Acad. Sci. USA 91:8132-8136 (1994) and Kim et al. J. Biol. Chem.269:24747-55 (1994)). When ErbB3 is co-expressed with ErbB2, an activesignaling complex is formed and antibodies directed against ErbB2 arecapable of disrupting this complex (Sliwkowski et al., J. Biol. Chem.,269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3 forheregulin (HRG) is increased to a higher affinity state whenco-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); Lewis, G. D. et al., Cancer Res., 56:1457-1465 (1996);Pinkas-Kramarski, R. et al. (1996) EMBO J. 15:2452-2467; Beerli, R. etal. (1995) Mol. Cell. Biol. 15:6496-6505; and Karunagaran, D. et al.(1996) EMBO J. 15:254-264 with respect to the in vivo ErbB2-ErbB3protein complex.

The class I subfamily of growth factor receptor protein tyrosine kinaseshas been further extended to include the HER4/Erb4 receptor. See EP PatAppln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA90:1746-1750 (1993); and Plowman et al., Nature 366:473-475 (1993).Plowman et al. found that increased HER4 expression closely correlatedwith certain carcinomas of epithelial origin, including breastadenocarcinomas. Diagnostic methods for detection of human neoplasticconditions (especially breast cancers) which evaluate HER4 expressionare described in EP Pat Appln No. 599,274.

The quest for the activator of the HER2 oncogene has lead to thediscovery of a family of heregulin polypeptides. These proteins appearto result from alternate splicing of a single gene which was mapped tothe short arm of human chromosome 8 by Lee, J. and Wood, W. I. (1993)Genomics 16:790-791).

Holmes et al. isolated and cloned a family of polypeptide activators forthe HER2 receptor which they called heregulin-α (HRG-α),heregulin-β1(HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like(HRG-β2-like), and heregulin-β3 (HRG-β3). See Holmes, W. E. et al.,Science 256:1205-1210 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060.The 45 kDa polypeptide, HRG-α, was purified from the conditioned mediumof the MDA-MB-231 human breast cancer cell line. These researchersdemonstrated the ability of the purified heregulin polypeptides toactivate tyrosine phosphorylation of the HER2 receptor in MCF7 breasttumor cells. Furthermore, the mitogenic activity of the heregulinpolypeptides on SK-BR-3 cells (which express high levels of the HER2receptor) was illustrated. Like other growth factors which belong to theEGF family, soluble HRG polypeptides appear to be derived from amembrane bound precursor (called pro-HRG) which is proteolyticallyprocessed to release the 45 kDa soluble form. These pro-HRGs lack aN-terminal signal peptide.

While heregulins are substantially identical in the first 213 amino acidresidues, they are classified into two major types, α and β, based ontwo variant EGF-like domains which differ in their C-terminal portions.Nevertheless, these EGF-like domains are identical in the spacing of sixcysteine residues contained therein. Based on an amino acid sequencecomparison, Holmes et al. found that between the first and sixthcysteines in the EGF-like domain, HRGs were 45% similar toheparin-binding EGF-like growth factor (HB-EGF), 35% identical toamphiregulin (AR), 32% identical to TGF-α, and 27% identical to EGF.

The 44 kDa neu differentiation factor (NDF), which is the rat equivalentof human HRG, was first described by Peles et al., Cell, 69:205-216(1992); and Wen et al., Cell, 69:559-572 (1992). Like the HRGpolypeptides, NDF has an immunoglobulin (Ig) homology domain followed byan EGF-like domain and lacks a N-terminal signal peptide. Subsequently,Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994) carried out“exhaustive cloning” to extend the family of NDFs. This work revealedsix distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmeset al., the NDFs are classified as either α or β polypeptides based onthe sequences of the EGF-like domains. Isoforms 1 to 4 are characterizedon the basis of the variable membrane stretch (between the EGF-likedomain and transmembrane domain). Also, isoforms a, b and c aredescribed which have variable length cytoplasmic domains. Theseresearchers conclude that different NDF isoforms are generated byalternative splicing and perform distinct tissue-specific functions. Seealso EP 505 148; WO 93/22424; and WO 94/28133 concerning NDF.

While the heregulin polypeptides were first identified based on theirability to activate the HER2 receptor (see Holmes et al., supra), it wasdiscovered that certain ovarian cells expressing neu and neu-transfectedfibroblasts did not bind or crosslink to NDF, nor did they respond toNDF to undergo tyrosine phosphorylation (Peles et al., EMBO J.12:961-971 (1993)). This indicated another cellular component wasnecessary for conferring full heregulin responsiveness. Carraway et al.subsequently demonstrated that ¹²⁵I-rHRGβ1₁₇₇₋₂₄₄ bound to NIH-3T3fibroblasts stably transfected with bovine erbB3 but not tonon-transfected parental cells. Accordingly, they conclude that ErbB3 isa receptor for HRG and mediates phosphorylation of intrinsic tyrosineresidues as well as phosphorylation of ErbB2 receptor in cells whichexpress both receptors. Carraway et al., J. Biol. Chem.269(19):14303-14306 (1994). Sliwkowski et al., J. Biol. Chem.269(20):14661-14665 (1994) found that cells transfected with HER3 aloneshow low affinities for heregulin, whereas cells transfected with bothHER2 and HER3 show higher affinities.

This observation correlates with the “receptor cross-talking” describedpreviously by Kokai et al., Cell 58:287-292 (1989); Stem et al., EMBO J.7:995-1001 (1988); and King et al., 4:13-18 (1989). These researchersfound that binding of EGF to the EGFR resulted in activation of the EGFRkinase domain and cross-phosphorylation of p185^(HER2). This is believedto be a result of ligand-induced receptor heterodimerization and theconcomitant cross-phosphorylation of the receptors within theheterodimer (Wada et al., Cell 61:1339-1347 (1990)).

Plowman and his colleagues have similarly studiedp180^(HER4)/p185^(HER2) activation. They expressed p180^(HER2) alone,p180^(HER4) alone, or the two receptors together in hunan T lymphocytesand demonstrated that heregulin is capable of stimulating tyrosinephosphorylation of p180^(HER4), but could only stimulate p180^(HER2)phosphorylation in cells expressing both receptors. Plowman et al.,Nature 336:473-475 (1993). Thus, heregulin is an example of a member ofthe EGF growth factor family that can interact with several receptors(Carraway and Cantley, Cell 78:5-8 (1994)). Additionally, the β-cellulinligand has been shown to bind to the EGF receptor and HER4, but does notbind HER3 (Riese II, D. J. et al. (1996) Oncogene 12:345-353).

The biological role of heregulin has been investigated by severalgroups. For example, Falls et al., (Cell 72:801-815 (1993)) found thatARIA plays a role in myotube differentiation, namely affecting thesynthesis and concentration of neurotransmitter receptors in thepostsynaptic muscle cells of motor neurons. Corfas and Fischbachdemonstrated that ARIA also increases the number of sodium channels inchick muscle. Corfas and Fischbach, J. Neuroscience, 13(5): 2118-2125(1993). It has also been shown that GGFII is mitogenic for subconfluentquiescent human myoblasts and that differentiation of clonal humanmyoblasts in the continuous presence of GGFII results in greater numbersof myotubes after six days of differentiation (Sklar et al., J. CellBiochem., Abst. W462, 18D, 540 (1994)). See also WO 94/26298 publishedNov. 24, 1994.

Holmes et al., supra, found that HRG exerted a mitogenic effect onmammary cell lines (such as SK-BR-3 and MCF-7). The mitogenic activityof GGFs on Schwann cells has also been reported. See, e.g., Brockes etal., J. Biol. Chem. 255(18):8374-8377 (1980); Lemke and Brockes, J.Neurosci. 4:75-83 (1984); Brockes et al., J. Neuroscience 4(1):75-83(1984); Brockes et al., Ann. Neurol. 20(3):317-322 (1986); Brockes, J.,Methods in Enzym., 147: 217-225 (1987) and Marchionni et al., supra.Schwann cells provide myelin sheathing around the axons of neurons,thereby forming individual nerve fibers. Thus, it is apparent thatSchwann cells play an important role in the development, function andregeneration of peripheral nerves. The implications of this from atherapeutic standpoint have been addressed by Levi et al., J.Neuroscience 14(3):1309-1319 (1994). Levi et al. discuss the potentialfor construction of a cellular prosthesis comprising human Schwann cellswhich could be transplanted into areas of damaged spinal cord. Methodsfor culturing Schwann cells ex vivo have been described. See WO 94/00140and Li et al., J. Neuroscience 16(6):2012-2019 (1996).

Pinkas-Kramarski et al. found that NDF seems to be expressed in neuronsand glial cells in embryonic and adult rat brain and primary cultures ofrat brain cells, and suggested that it may act as a survival andmaturation factor for astrocytes (Pinkas-Kramarski et al., PNAS, USA91:9387-9391 (1994)). Meyer and Birchmeier, PNAS, USA 91:1064-1068(1994) analyzed expression of heregulin during mouse embryogenesis andin the perinatal animal using in situ hybridization and RNase protectionexperiments. These authors conclude that, based on expression of thismolecule, heregulin plays a role in vivo as a mesenchymal and neuronalfactor. Also, their findings imply that heregulin functions in thedevelopment of epithelia. Similarly, Danilenko et al., Abstract 3101,FASEB 8(4-5):A535 (1994), found that the interaction of NDF and the HER2receptor is important in directing epidermal migration anddifferentiation during wound repair.

Interaction of ErbB family members has been investigated in vitro and invivo. Transactivation of ErbB2 as a result of ligand interaction withother ErbB family members is a common and physiologically importantoccurrence (Dougall, W. C. et al., (1993) J. Cell. Biochem. 53:61-73;Earp, H. S. et al., (1995) Breast Cancer Res. Treatment 35:115-132).Co-expression of ErbB2 with ErbB3 leads to the formation of a highaffinity heregulin (HRG) binding site (Sliwkowski, M. X. et al., (1994)J. Biol. Chem. 269:14661-14665). ErbB2 modulates the affinity of ErbB3for HRG and appears to provide tyrosine kinase activity to the ErbB3-HRGcomplex, since ErbB3 is a dysfunctional signaling receptor lackingintrinsic tyrosine kinase activity (Guy, P. M. et al, (1994) PNAS USA91:8132-8136). Physio-chemical studies have not shown association of theECDs of ErbB2 and ErbB3 in vitro (Horan et al., J. Biol. Chem.270:24604-24608 (1995)). In addition, binding of neu differentiationfactor (NDF) to soluble HER3 was not enhanced by the presence of solubleHER2.

SUMMARY OF THE INVENTION

The invention relates to the surprising discovery that soluble chimericheteromultimers comprising the extracellular domains of aheteromultimeric receptor monomers bind the receptor ligand. Theinvention further relates to methods of making the chimericheteromultimers, methods of using them as receptor ligand antagonists,antibodies to the chimeric heteromultimers that function as antagonistsor agonists of the receptor ligand, and methods of treating a diseasestate related to ligand-receptor interaction.

In one aspect the invention includes a chimeric heteromultimercomprising a first amino acid sequence, which sequence forms a chimericmonomer and comprises an extracellular domain (ECD) or ligand bindingfragment thereof, of a first monomer of a natural heteromultimericreceptor and a multimerization domain, wherein the ECD is fused to themultimerization domain. The chimeric heteroadhesin of the inventionfurther comprises an additional acid sequence forming an additionalchimeric monomer comprising an extracellular domain of an additionalmonomer of the natural heteromultimeric receptor and a multimerizationdomain. According to this aspect of the invention the extracellulardomains of the first and additional monomers of the naturalheteromultimeric receptor are associated in a cell to form a naturalheteromultimeric receptor which is activated upon binding of a ligand,and

wherein the soluble chimeric heteromultimer adhesin has 10⁻¹ to 10⁶ foldaffinity for the ligand relative to a monomer of the natural receptor ora homomultimer of the natural receptor. In a preferred embodiment of theinvention the chimeric heteromultimer adhesin is an aqueous solubleadhesin.

In an embodiment of the invention, the chimeric heteromultimer adhesinis an antagonist of the ligand that binds to the extracellular domainsof the natural heteromultimeric receptor.

In another embodiment of the invention, the multimerization domain ofthe first amino acid sequence is capable of interacting with themultimerization domain of each additional amino acid sequence to form aheteromultimer.

In yet another embodiment of the invention the chimeric heteromultimeradhesin comprises a multimerization domain which includes animmunoglobulin region, preferably an immunoglobulin constant region,such as from IgG1, IgG2, IgG3, IgG4, IgM, and IgF.

In still another embodiment of the invention, the chimeric heteroadhesinincludes a multimerization domain capable of forming a stableprotein-protein interaction. Such protein-protein interaction domains(or multimerization domains) include a leucine zipper, an amino acidsequence comprising a protuberance complementary to an amino acidsequence comprising a hole, a hydrophobic domain, a hydrophilic domain,and an amino acid sequence comprising a free thiol moiety capable ofreacting to form an intermolecular disulfide bond with a multimerizationdomain of an additional amino acid sequence.

A further embodiment of the invention is a chimeric heteromultimeradhesin in which the first amino acid sequence comprising anextracellular domain of the ErbB2 receptor monomer, an additional aminoacid sequence comprising an extracellular domain of the ErbB3 receptormonomer, and a multimerization domain of the first and additional aminoacids each comprises an immunoglobulin constant region. Themultimerization domain provides for the formation of a stableprotein-protein interaction between the first and additional amino acidsequences. A preferred ligand of this chimeric heteroadhesin is theligand, heregulin.

A further embodiment of the invention is a chimeric heteromultimeradhesin in which the first amino acid sequence comprising anextracellular domain of the ErbB2 receptor monomer, an additional aminoacid sequence comprising an extracellular domain of the ErbB4 receptormonomer, and a multimerization domain of the first and additional aminoacids each comprises an immunoglobulin constant region. Themultimerization domain provides for the formation of a stableprotein-protein interaction between the first and additional amino acidsequences. A preferred ligand of this chimeric heteroadhesin is theligand, heregulin.

In another aspect, the invention includes an isolated nucleic acidsequence encoding an amino acid sequence of the chimeric heteromultimeradhesin of the invention.

In other embodiments, the invention provides an isolated nucleic acidmolecule encoding the chimeric amino acid sequence of a monomer of theheteromultimer adhesin such as, for example, ErbB2-IgG, ErbB3-IgG, orErbB4-IgG. For example, the nucleic acid molecule may be selected fromthe group consisting of: (a) a nucleic acid comprising the nucleotidesequence of the extracellular domain (i.e. a ligand binding domain orbinding fragment thereof) of a monomer of a natural heteromultimericreceptor covalently attached in phase and in the direction oftranscription to a nucleic acid sequence encoding a multimerizationdomain, such as an immunoglobulin constant domain; and (b) a nucleicacid corresponding to the sequence of (a) within the scope of degeneracyof the genetic code. The isolated nucleic acid molecule optionallyfurther comprises a promoter operably linked thereto.

The isolated nucleic acid may also be used for in vivo or ex vivo genetherapy. This embodiment of the invention encompasses the administrationof the nucleic acid of the invention, a vector comprising the nucleicacid, or a cell comprising the nucleic acid to a mammal such that theencoded chimeric adhesin is expressed in the mammal and acts as anantagonist of its ligand. For example, ErbB2/3-IgG expressed in a mammalis useful to reduce the local concentration of heregulin near a ErbB2/3receptor and inhibit growth of a cell having the receptor on itssurface. Preferably the expressed ErbB2/3-IgG is used to treat a cellproliferative disease, such as a cancer, in which antagonizing heregulinbinding to its receptor inhibits cell growth.

In an embodiment of the invention, the isolated nucleic acid sequence ofthe chimeric amino acid encodes an extracellular domain or bindingfragment thereof from the ErbB2 receptor, and wherein themultimerization domain comprises an immunoglobulin constant region.

In still another embodiment of the invention, the isolated nucleic acidsequence of the chimeric amino acid sequence encodes an extracellulardomain or binding fragment thereof from the ErbB3 receptor ECD, andwherein the multimerization domain comprises an immunoglobulin constantregion.

In another embodiment of the invention, the isolated nucleic acidsequence of the chimeric aminio acid sequence encodes an extracellulardomain or binding fragment thereof from the ErbB4 receptor ECD, andwherein the multimerization domain comprises an immunoglobulin constantregion.

Another embodiment of the invention includes a promoter operably linkedto the nucleic acid molecule.

In still another embodiment, the invention includes a vector comprisingthe isolated nucleic acid of the invention. For example, the inventionprovides a vector comprising the nucleic acid molecule (e.g. anexpression vector comprising the nucleic acid molecule operably linkedto control sequences recognized by a host cell transformed with thevector); a host cell comprising the nucleic acid molecule; and a methodof using a nucleic acid molecule encoding a chimeric heteromultimeradhesin, such as an ErbB-IgG, to effect production of the adhesin whichcomprises the step of culturing the host cell and recovering the adhesinfrom the cell culture. In a related embodiment the method of using thenucleic acid to effect production of the adhesin includes introducingmultiple nucleic acid sequences encoding different chimeric adhesins andexpressing a mixture of chimeric adhesins. For example, a nucleic acidencoding ErbB2-IgG and a nucleic acid encoding ErbB3-IgG are introducedinto a host cell, expressed, and a mixture of the homodimers andheterodimer is isolated from the cell or from the culture medium.

An embodiment of the invention further includes a host cell comprisingthe nucleic acid of the invention. Preferably the host cell is capableof expressing the nucleic acid, which expression includes thetranslation and production of the chimeric heteroadhesin of theinvention. The embodiment of the invention encompasses a host cellcomprising and expressing a chimeric monomer of the heteroadhesin, whilein another host cell of the invention an additional chimeric monomer ofthe heteroadhesin is expressed. Alternatively, the embodimentencompasses the expression of more than one chimeric monomer in a singlehost cell.

In a preferred embodiment of the invention, the host cell comprises afirst isolated nucleic acid sequence encoding the first amino acidsequence of the soluble chimeric heteromultimer of the invention,wherein the extracellular domain is from the ErbB2 receptor and whereinthe multimerization domain comprises an immunoglobulin constant region;and a second isolated nucleic acid sequence encoding an additional aminoacid sequence of the soluble chimeric heteromultimer of the invention,wherein the extracellular domain is from the ErbB3 receptor and whereinthe multimerization domain comprises an immunoglobulin constant region.

In another preferred embodiment of the invention, the host cellcomprises a first isolated nucleic acid sequence encoding the firstamino acid sequence of the soluble chimeric heteromultimer of theinvention, wherein the extracellular domain is from the ErbB2 receptorand wherein the multimerization domain comprises an immunoglobulinconstant region; and a second isolated nucleic acid sequence encoding anadditional amino acid sequence of the soluble chimeric heteromultimer ofthe invention, wherein the extracellular domain is from the ErbB4receptor and wherein the multimerization domain comprises animmunoglobulin constant region.

Another aspect of the invention includes an antagonist antibody to thechimeric heteromultimer adhesin of the invention, wherein the antibodybinds to the natural heteromultimeric receptor and inhibits itsactivation.

Another aspect of the invention includes an agonist antibody to thechimeric heteromultimer adhesin of the invention, wherein the antibodybinds to the natural heteromultimeric receptor and activates it. Inpreferred embodiments of the invention, the agonist antibody is capableof activating the natural heteromultimeric receptor at 10⁻¹ to 10⁶ foldthe activity of the natural ligand.

The chimeric heteromultimer-specific antibodies may be used, among otherthings, in a method for detecting heteromultimeric receptors whichcomprises the step of contacting a sample suspected of containing theheteromultimeric receptor with the antibody (which is optionallylabeled) and detecting if binding has occurred. The antibody may also beused in a method for purifying the heteromultimeric receptor whichcomprises the step of passing a mixture containing the heteromultimericreceptor over a solid phase to which is bound the antibody andrecovering the fraction containing the heteromultimeric receptor.Preferably, in one embodiment of the invention the heteromultimericreceptor is ErbB2/ErbB4 and the chimeric heteromultimer adhesin isErbB2-IgG/ErbB4-IgG. In another preferred embodiment, theheteromultimeric receptor is ErbB2/ErbB3 and the chimeric heteromultimeradhesin is ErbB2-IgG/ErbB3-IgG.

In yet another aspect, the invention includes a method of forming achimeric heteromultimer adhesin-ligand complex in a sample comprisingthe ligand. The method of the invention includes contacting the chimericheteromultimer adhesin of the invention with the sample under conditionssuch that the ligand binds to the heteromultimer to form a chimericheterodimer adhesin-ligand complex.

In an embodiment of the invention, the chimeric heteromultimeradhesin-ligand complex inhibits binding of the ligand to the naturalheteromultimer receptor. Preferably the sample is a mammalian tissue ora mammalian fluid, such as a body fluid including, but not limited toblood, serum, plasma, lymph, and urine. Preferably, the mammal is ahuman.

In another aspect, the invention involves a method of inhibiting naturalheteromultimer receptor activation. The method includes the steps of 1)contacting the chimeric heteromultimer adhesin of the invention with asample containing a ligand for the natural heteromultimeric receptor andthe receptor; and 2) incubating the chimeric heteromultimer adhesin withthe ligand to form a complex such that activation of the naturalheteromultimeric receptor by the ligand is inhibited.

In an embodiment of the method of inhibiting ligand binding to a naturalheteromultimer receptor, the natural heteromultimeric receptor is ErbBand the soluble chimeric heteromultimer comprises the extracellulardomains of ErbB2 and ErbB3.

In another embodiment of the method of inhibiting ligand binding to anatural heteromultimer receptor, the natural heteromultimeric receptoris ErbB and the soluble chimeric heteromultimer comprises theextracellular domains of ErbB2 and ErbB4.

Another embodiment of the invention is a method of inhibiting ligandbinding to a natural heteromultimer receptor, wherein receptoractivation is inhibited. The method comprises contacting the antagonistantibody of the invention with the natural heteromultimeric receptor toform an antagonist antibody-heteromultimer receptor complex, whereinactivation of the receptor is inhibited.

In another aspect, the invention involves a method of activating anatural heteromultimeric receptor comprising contacting the agonistantibody of the invention with the natural heteromultimeric receptor toform agonist antibody-heteromultimeric receptor complex, wherein thereceptor is activated.

In still another aspect, the invention involves a method for thetreatment of a disease state comprising administering to a mammal inneed thereof a therapeutically effective dose of the chimericheteromultimer adhesin of the invention. Embodiments of the inventionencompass disease states in which the disease is treatable by inhibitingcontact between the ligand and the natural heteromultimeric receptorsuch as by competitive binding of the heteroadhesin to the ligand.

In an embodiment of the invention, the chimeric heteromultimer adhesinis an ErbB2/ErbB3-Ig heteroadhesin. In another embodiment, the chimericheteromultimer is an ErbB2/ErbB4-Ig heteroadhesin.

The invention encompasses a composition comprising the chimericheteromultimer adhesin. The composition comprising the adhesin ispreferably sterile. Where the composition is an aqueous solution,preferably the adhesin is soluble. Where the composition is alyophilized powder, preferably the powder is reconstitutable in anappropriate solvent.

In another embodiment of the invention, the treatment method comprisesadministering chimeric heteromultimer adhesins which comprise chimericmonomers, each prepared using an extracellular domain of theheteromultimeric receptor monomers of interest. The extracellulardomains are preferably from receptors selected from the followingheteromultimeric receptors: Axl, Rse, epidermal grouth factor (EGF)receptor, hepatocyte growth factor (HGF) receptor, IL-2, c-mer, Al-1,EPH, TrkA, TrkB, TrkC, TNF, IL-10, CRF2-4, RXR, RON, AChRα/δ, TRα/RXRα,Trα/DR4, Trα/MHC-TRE, Trα/ME, Trα/F2, KDR/FLT-1, FLT/VEGF, VEGF121/165,Arnt/Ahr, CGA/CGB, EGFR/p185-neu, prolactin receptor (PRL), T cellreceptor (TCR), fibroblast growth factor (FGF), and Cak receptor(Kishimoto, T. et al. (1994) Cell 76:253-262; Kendall, R. L., et al.(1996) Biochem Biophys. Res. Comm. 226:324-328; Chang, W.-P. AndClevenger, C. V. (1996) PNAS USA 93:5947-5952; Lala, D. S. et al. (1996)Nature 383:450-453; Collesi, C. et al. (1996) Mol. Cell. Biol.16:5518-5526; Tzahar, E. et al. (1996) Mol. Cell. Biol. 16:5276-5287;Shtrom, S. S. and Hall, Z. W. (1996) J. Biol. Chem. 271:25506-25514;Nagaya, T. et al. (1996) Biochem. Biophys. Res. Comm. 226:426-430;Dendall, R. L. et al. (1996) Biochem. Biophys. Res. Comm. 226:324-328;Kainu, T. et al. (1995) Neuroreport 6:2557-2560; Yoo, S. H. and Lewis,M. S. (1996) J. Biol. Chem. 271:17041-17046; Murali, R. et al. (1996)PNAS USA 93:6252-6257; Dietrich J. et al. (1996) J. Cell Biol.132:299-310; Tanahashi, T. et al. (1996) J. Biol. Chem 271:8221-8227;and Perez, J. L. et al. (1996) Oncogene 12:1469-1477). The extracellulardomains are more preferably from receptors selected from the following:IL-6/gp130, IL-11/gp130 leukemia inhibitory factor (LIF)/gp130,cardiotrophin-1/gp130 (CT-1), IL-11/gp130, ciliary neurotrophic factorCNTF/gp130, oncostatin M (OSM)/gp130, interferon γ, and interferon α,β(Kishimoto, T. et al. (1994), supra; Taga, T. (1996) J. Neurochem.67:1-10; Pennica, D. et al. (1995) J. Biol. Chem. 270:10915-10922;Marsters, S. A. (1995) PNAS USA 92:5401-5405; and Wollert, K. C. et al.(1996) J. Biol. Chem. 271:9535-9545). Most preferably, the extracellulardomains are selected from the ErbB family of receptors.

Embodiments of the method of treatment encompass a disease state orstates such as immunological disorders, cancer, and neurologicaldisorder.

In embodiments where the heteroadhesin is an ErbB2/ErbB3-Ig or anErbB2/ErbB4-lg heteroadhesin, the method of treatment encompasses adisease state selected from the group consisting of inflammatorydisease, cancer, neurological disorders such as neurofibromatosis andperipheral neuropathy, and cardiac disorders such as cardiachypertrophy.

The invention further provides a method for treating a mammal comprisingadministering a therapeutically effective amount of a chimericheteromultimer adhesin, such as ErbB2/3-IgG or ErbB2/4-IgG to themammal. For example, the mammal may be suffering from a neurologicaldisorder or cell proliferative disease. The mammal is one which couldbenefit from a reduction in HRG levels/biological activity (e.g. incancer).

In another aspect, the invention includes pharmaceutical compositions.In an embodiment of the invention the pharmaceutical compositioncomprises a chimeric heteromultimer adhesin of the invention, whichheteroadhesin 1) comprises an ECD or binding fragment thereof of anatural heteromultimeric receptor, and 2) is an antagonist of the ligandwhich binds the ECD of the natural heteromultimeric receptor.

In another embodiment of the invention the pharmaceutical compositioncomprises an antibody to a chimeric heteromultimer adhesin of theinvention, which anti-heteroadhesin antibody 1) comprises an ECD orbinding fragment thereof of a natural heteromultimeric receptor, and 2)binds to the ECD of the natural heteromultimeric receptor and is anantagonist of the ligand which binds the ECD of the naturalheteromultimeric receptor.

In still another embodiment of the invention the pharmaceuticalcomposition comprises an antibody to a chimeric heteromultimer adhesinof the invention, which anti-heteroadhesin antibody 1) comprises and ECDor binding fragment thereof of a natural heteromultimeric receptor, and2) binds to the ECD of the natural heteromultimeric receptor and is anagonist of the ligand which binds the ECD of the naturalheteromultimeric receptor.

In yet another aspect, the invention includes articles of manufacturecomprising a container, a label on the container, and a compositioncontained within the container. In one embodiment of the invention, thecomposition comprises the chimeric heteromultimer adhesin composition ofthe invention, which heteroadhesin is an antagonist of ligand. Thecomposition is effective for antagonizing binding of the ligand to itsnatural heteromultimeric receptor, and the label on the containerindicates that the composition can be used for antagonizing binding ofthe ligand to the natural heteromultimeric receptor. In a preferredembodiment the chimeric heteromultimer adhesin is selected from thegroup consisting of ErbB2/ErbB3-Ig or ErbB2/ErbB4-Ig.

In another embodiment of the article of manufacture, the compositioncomprises an anti-chimeric heteromultimer adhesin antibody, whichantibody is an antagonist of a ligand. The composition is effective forantagonizing binding of the ligand to its natural heteromultimericreceptor, and the label on the container indicates that the compositioncan be used for antagonizing binding of the ligand to the naturalheteromultimeric receptor. In a preferred embodiment the anti-chimericheteromultimer adhesin antibody is an antibody raised to a chimericheteroadhesin selected from the group consisting of ErbB2/ErbB3-Ig orErbB2/ErbB4-Ig.

In yet another embodiment of the article of manufacture, the compositioncomprises an anti-chimeric heteromultimer adhesin antibody, whichantibody is an agonist of a ligand. The composition is effective foractivating the natural heteromultimeric receptor of the ligand, and thelabel on the container indicates that the composition can be used foractivating the natural heteromultimeric receptor. In a preferredembodiment the anti-chimeric heteromultimer adhesin antibody is anantibody raised to a chimeric heteroadhesin selected from the groupconsisting of ErbB2/ErbB3-Ig or ErbB2/ErbB4-Ig.

These and other aspects of the invention will be apparent to thoseskilled in the art upon consideration of the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the ErbB family of chimeric homodimers andheterodimers. The extracellular domains (ECD) and immunoglobulin region(Fc) of the chimeras are indicated. The extracellular domains arederived from the natural heteromultimeric receptor and are fused byrecombinant means to a multimerization domain, the immunoglobulinregion.

FIG. 2 is a graphical plot showing the binding analysis of the chimericimmunoadhesin. The homodimeric ErbB3-IgG and ErbB4-IgG were capable ofspecifically binding ¹²⁵I-HRG, whereas no discernible binding wasdetected with the ErbB2-IgG construct.

FIG. 3A-3D are graphical results of ¹²⁵I-heregulin binding studies foreach of the chimeric heteroadhesins ErbB2/3-IgG, ErbB2/4-IgG andErbB3/4-IgG. As shown in FIG. 3A, a high affinity HRG binding site couldbe detected with the ErbB2-containing heterodimers but not theErbB3/4-IgG.

FIG. 4A and 4B are graphical results of anti-ErbB2 monoclonal antibody(2C4) binding studies in which the binding activity of chimeric ErbBhomodimers is compared to that of chimeric ErbB heterodimers in thepresence of 2C4.

FIG. 5 is a bar graph indicating the ability of the ErbB-IgG proteins toinhibit HRG-dependent thymidine incorporation in the breast carcinomacell line, MCF7. Varying concentrations of the different ErbB-IgGproteins were incubated with 1 nM rHRG and then added to serum-starvedmonolayer cultures of MCF7 cells. Cells were labeled with ³H-thymidineto measure DNA synthesis. Receptor fusions capable of HRG bindinginhibited the HRG-mediated mitogenic response in a dose related manner.The heterodimeric IgGs, ErbB3/2-IgG and ErbB4/2-IgG, were more potentthan their corresponding homodimeric fusion proteins

FIG. 6 is a diagram depicting possible models for the interaction ofErbB2 with ErbB3 or ErbB4, a “contact” model (left) and a“conformational” model (right).

DETAILED DESCRIPTION

Before the present chimeric heteromultimer adhesins, methods of makingthem, and uses therefor are described, it is to be understood that thisinvention is not limited to the particular adhesins or processesdescribed as such compounds and methods may, of course, vary. It is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting since the scope of the present invention will be limited onlyby the appended claims.

I. Definitions

In general, the following words or phrases have the indicated definitionwhen used in the description, examples, and claims.

Unless indicated otherwise, the term “ErbB” when used herein refers toany one or more of the mammalian ErbB receptors (ie. ErbB1 or epidermalgrowth factor (EGF) receptor; ErbB2 or HER2 receptor, ErbB3 or HER3receptor; ErbB4 or HER4 receptor, and any other member(s) of this classI tyrosine kinase family to be identified in the future) and “erbB”refers to the mammalian erbB genes encoding these receptors.

“HRG” (or “heregulin”) is defined herein to be any polypeptide sequencethat possesses at least one biological property (as defined below) ofnative sequence HRG (U.S. Application Ser. No. 60/021,640, PR1043,supra). This definition encompasses not only the polypeptide isolatedfrom a native HRG source such as human MDA-MB-175 cells or from anothersource, such as another animal species, but also the polypeptideprepared by recombinant or synthetic methods. It also includes variantforms including functional derivatives, allelic variants, naturallyoccurring isoforms and analogues thereof. Sometimes the HRG is “nativeHRG” which refers to endogenous HRG polypeptide which has been isolatedfrom a mammal. The HRG can also be “native sequence HRG” insofar as ithas the same amino acid sequence as a native HRG (e.g. human HRG).However, “native sequence HRG” encompasses the polypeptide produced byrecombinant or synthetic means. “Mature HRG” is soluble or secreted HRGreleased from the cell (i.e. lacking amino-terminal sequence). HRG“isoforms” are naturally occurring polypeptides which comprise at leastpart of the N-terminal domain of HRG.

The term “immunoadhesin” as used herein refers to antibody-likemolecules which combine the binding domain of a protein such as anextracellular domain (the adhesin portion) of a cell-surface receptorwith the effector functions of an immunoglobulin constant domain.Immunoadhesins can possess many of the valuable chemical and biologicalproperties of human antibodies. Since immunoadhesins can be constructedfrom a human protein sequence with a desired specificity linked to anappropriate human immunoglobulin hinge and constant domain (Fc)sequence, the binding specificity of interest can be achieved usingentirely human components. Such immunoadhesins are minimally immunogenicto the patient, and are safe for chronic or repeated use.

Immunoadhesins reported in the literature include fusions of the T cellreceptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84:2936-2940(1987)); CD4 (Capon et al., Nature 337:525-531 (1989); Traunecker etal., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin or homing receptor (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp.Med. 174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); NPreceptors (Bennett et al., J. Biol. Chem. 266:23060-23067 (1991));inteferon γ receptor (Kurschner et al., J. Biol. Chem. 267:9354-9360(1992)); 4-1BB (Chalupny et al., PNAS USA 89:10360-10364 (1992)) and IgEreceptor α (Ridgway and Gorman, J. Cell. Biol. 115, Abstract No. 1448(1991)).

Examples of homomultimeric immunoadhesins which have been described fortherapeutic use include the CD4-IgG immunoadhesin for blocking thebinding of HIV to cell-surface CD4. Data obtained from Phase I clinicaltrials in which CD4-IgG was administered to pregnant women just beforedelivery suggests that this immunoadhesin may be useful in theprevention of maternal-fetal transfer of HIV. Ashkenazi et al., Intern.Rev. Immunol. 10:219-227 (1993). An immunoadhesin which binds tumornecrosis factor (TNF) has also been developed. TNF is a proinflammatorycytokine which has been shown to be a major mediator of septic shock.Based on a mouse model of septic shock, a TNF receptor immunoadhesin hasshown promise as a candidate for clinical use in treating septic shock(Ashkenazi, A. et al. (1991) PNAS USA 88:10535-10539). Immunoadhesinsalso have non-therapeutic uses. For example, the L-selectin receptorimmunoadhesin was used as a reagent for histochemical staining ofperipheral lymph node high endothelial venules (HEV). This reagent wasalso used to isolate and characterize the L-selectin ligand (Ashkenaziet al., supra).

If the two arms of the immunoadhesin structure have differentspecificities, the immunoadhesin is called a “bispecific immunoadhesin”by analogy to bispecific antibodies. Dietsch et al., J. immunol. Methods162:123 (1993) describe such a bispecific immunoadhesin combining theextracellular domains of the adhesion molecules, E-selectin andP-selectin, each of which selectins is expressed in a different celltype in nature. Binding studies indicated that the bispecificimmunoglobulin fusion protein so formed had an enhanced ability to bindto a myeloid cell line compared to the monospecific immunoadhesins fromwhich it was derived.

The term “heteroadhesin” is used interchangeably with the expression“chimeric heteromultimer adhesin” and refers to a complex of chimericmolecules (amino acid sequences) in which each chimeric moleculecombines a biologically active portion, such as the extracellular domainof each of the heteromultimeric receptor monomers, with amultimerization domain. The “multimerization domain” promotes stableinteraction of the chimeric molecules within the heteromultimer complex.The multimerization domains may interact via an immunoglobulin sequence,leucine zipper, a hydrophobic region, a hydrophilic region, or a freethiol which forms an intermolecular disulfide bond between the chimericmolecules of the chimeric heteromultimer. The multimerization domain maycomprise an immunoglobulin constant region. A possible multimerizationdomain useful in the present invention is found in U.S. application Ser.No. 07/440,625, P565P1 (herein incorporated by reference) in whichhybrid immunoglobulins are described. In addition a multimerizationregion may be engineered such that steric interactions not only promotestable interaction, but further promote the formation of heterodimersover homodimers from a mixture of monomers. See, for example, U.S.application Ser. No. 08/399,106, P0927 (herein incorporated by referencein its entirety) in which a “protuberance-into-cavity” strategy isdisclosed for an interface between a first and second polypeptide forhetero-oligomerization. “Protuberances” are constructed by replacingsmall amino acid side chains from the interface of the first polypeptidewith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the protuberances areoptionally created on the interface of the second polypeptide byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). The immunoglobulin sequence preferably, but notnecessarily, is an immunoglobulin constant domain. The immunoglobulinmoiety in the chimeras of the present invention may be obtained fromIgG₁, IgG₂, IgG₃ or IgG₄ subtypes, IgA, IgE, IgD or IgM, but preferablyIgG₁ or IgG₃. The term “epitope tagged” when used herein refers to achimeric polypeptide comprising the entire chimeric heteroadhesin, or afragment thereof, fused to a “tag polypeptide”. The tag polypeptide hasenough residues to provide an epitope against which an antibody can bemade, yet is short enough such that it does not interfere with activityof the chimeric heteroadhesin. The tag polypeptide preferably is fairlyunique so that the antibody thereagainst does not substantiallycross-react with other epitopes. Suitable tag polypeptides generallyhave at least 6 amino acid residues and usually between about 8-50 aminoacid residues (preferably between about 9-30 residues). An embodiment ofthe invention encompasses a chimeric heteroadhesin linked to an epitopetag, which tag is used to detect the adhesin in a sample or recover theadhesin from a sample.

“Isolated chimeric heteromultimer adhesin”, “highly purified chimericheteromultimer adhesin” and “substantially homogeneous chimericheteromultimer adhesin” are used interchangeably and mean the adhesinthat has been purified from a source or has been prepared by recombinantor synthetic methods and is sufficiently free of other peptides orproteins to homogeneity by chromatographic techniques or otherpurification techniques, such as SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins. As disclosed herein (below), the ErbB2/3-IgG orErbB2/4-IgG chimeric heteroadhesins of the invention bind withsufficiently greater affinity relative to the homodimers that the use ofa mixture of homodimers and heterodimers is also considered a usefulembodiment of the invention. The terms “chimeric heteromultimeradhesin”, “chimeric heteroadhesin” and “CHA” are used interchangeablyherein.

“Biological property” when used in conjunction with “chimericheteromultimer adhesin” means an ability to bind a ligand and functionas an antagonist of the ligand for binding to the natural receptor.“Biological property” when used in conjunction with “an antibody to achimeric heteromultimer adhesin” means an ability to bind theextracellular domains encoded in the adhesin or the extracellulardomains of the natural heteromultimeric receptor such that the antibodyacts as an antagonist or an agonist of the ligand.

“Biological activity” where used in conjunction with a chimericheteroadhesin such as the ErbB heteroadhesins includes functioning as anantagonist of heregulin receptor activation (e.g. antagonizingactivation of the ErbB2, ErbB3 and/or ErbB4 receptor) by binding to cellmembrane associated heregulin or secreted heregulin; inhibition ofgrowth of cells expressing ErbB receptors on their surface; inhibitionof differentiation and/or proliferation of cells expressing thesereceptors (e.g. SK-BR-3 cells, Schwann cells, hepatocytes, glioblastomacells, epithelial cells (such as in breast, ovary, prostate, lung,pancreas, colon and rectum), muscle cells, astrocytes and/oroligodendrocytes); inhibition of receptor binding (e.g. to the ErbB2/3,ErbB2/4, ErbB3 and/or ErbB4 receptor); inhibition of mitogenic activity;inhibiting acetylcholine receptor synthesis at the neuromuscularjunction; and inhibiting formation of a synaptic junction between aneuron and a muscle, nerve or glandular cell.

“Biological activity” where used in conjunction with an agonist chimericheteroadhesin antibody such as an agonist anti-ErbB heteroadhesinsantibody include functioning as an agonist of heregulin receptoractivation (e.g. activation of the ErbB2, ErbB3 and/or ErbB4 receptor);receptor binding and activation(e.g. to the ErbB2/3, ErbB2/4, ErbB3and/or ErbB4 receptor); promoting growth of cells expressing ErbBreceptors on their surface; promoting differentiation and/orproliferation of cells expressing these receptors (e.g. SK-BR-3 cells,Schwann cells, hepatocytes, glioblastoma cells, epithelial cells (suchas in breast, ovary, prostate, lung, pancreas, colon and rectum), musclecells, astrocytes and/or oligodendrocytes); promoting mitogenicactivity; promoting acetylcholine receptor synthesis at theneuromuscular junction; and promoting formation of a synaptic junctionbetween a neuron and a muscle, nerve or glandular cell.

“Percent amino acid sequence identity” with respect to the chimericheteromultimer adhesin is defined herein as the percentage of amino acidresidues in the candidate extracellular domain sequence that areidentical with the residues in the extracellular domain sequence of amonomer of the natural heteromultimeric receptor, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theadhesin sequence shall be construed as affecting sequence identity orhomology.

The term “disease state” refers to a physiological state of a cell or ofa whole mammal in which an interuption, cessation, or disorder ofcellular or body functions systems, or organs has occurred.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastric cancer,pancreatic cancer, glial cell tumors such as glioblastoma andneurofibromatosis, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer. Where the chimericheteroadhesin of the invention in an ErbB-Ig heteroadhesin, the cancerto be treated is preferably cancerous growth of cells expressin the ErbBreceptors, such as cancerous growth of breast, ovary, prostate, lung,pancreas, and colorectal cells.

The term “inflammatory disorder” refers to a fundamental pathologicprocess consisting of a dynamic complex of cytologic and histologicreactions that occur in the affected blood vessels and adjacent tissuesin response to an injury or abnormal stimulation caused by a physical,chemical, or biologic agent, including: 1) the local reactions andresulting morphologic changes, 2) the destruction or removal of theinjurious material, 3) the responses that lead to repair and healing.Inflammatory disorders treatable by the invention are those wherein theinflammation is associated with cytokine-induced disorders, such asthose associated with interleukin and leukemia inhibitory factorcytokines. Such disorders include abnormalities in thrombopoiesis,macrophage growth and differentiation, proliferation of hematopoieticprogenitors, and the like.

The term “neurological disorder” refers to or describes thephysiological condition in mammals that is typically characterized bynerve cell growth, differentiation, or cell signalling. Examples ofneurological disorders include, but are not limited to,neurofibromatosis and peripheral neuropathy.

The term “cardiac disorder” refers to or describes the physiologicalcondition in mammals that is typically characterized by cardiac cellgrowth and differentiation. An example of a cardiac disorder includes,but is not limited to, cardiac hypertrophy and heart failure, includingcongestive heart failure, myocardial infarction, and tachyarrhythmia.“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues.

“Determining disease status” refers to the act of determining likelihoodof patient survival and time to relapse for neoplastic diseases,particularly breast, ovarian, stomach, endometrial, salivary gland,lung, kidney, colon, and bladder carcinomas. In particular, an antibodyof the invention (raised to the chimeric heteroadhesin of the inventionand capable of interacting with the extracellular domains) can be usedto quantify the heteromultimeric receptor (e.g., ErbB2, ErbB3 or ErbB4,but normally ErbB2) overexpression in cancerous tissue taken from apatient suffering from carcinoma. This can also be referred to as“determining the proper course of treatment for patients suffering fromcancer”. For example, those patients characterized by ErbB2overexpression or having increased amounts of ErbB2/3 or ErbB2/4 cellsurface receptors may require more aggressive treatment (e.g. high dosesof chemo- or radiotherapy treatment) than might otherwise be indicatedby other diagnostic factors. This phrase encompasses diagnosing patientssuffering from high grade ductal carcinoma in situ, including extensiveintraductal carcinoma. See, e.g., Disis et al., Cancer Research,54:16-20 (1994).

The word “sample” refers to tissue, body fluid, or a cell from apatient. Normally, the tissue or cell will be removed from the patient,but in vivo diagnosis is also contemplated. In the case of a solidtumor, a tissue sample can be taken from a surgically removed tumor andprepared for testing by conventional techniques. In the case oflymphomas and leukemias, lymphocytes, leukemic cells, or lymph tissueswill be obtained and appropriately prepared. Other patient samples,including urine, tear drops, serum, cerebrospinal fluid, feces, sputum,cell extracts etc will also be useful for particular tumors.

The expression “labeled” when used herein refers to a molecule (e.g. achimeric heteroadhesin such as ErbB2/3-IgG) which has been conjugated,directly or indirectly, with a detectable compound or composition. Thelabel may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzea chemical alteration of a substrate compound or composition which isdetectable.

By “solid phase” is meant a non-aqueous matrix to which a reagent ofinterest (e.g., ErbB2/3-IgG, ErbB2/4-IgG or an antibody thereto) canadhere. Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g.,controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g.,an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

The phrase “activating an ErbB receptor” refers to the act of causingthe intracellular kinase domain of an ErbB receptor to phosphorylatetyrosine residues. Generally, this will involve binding of an antibodyraised to ErbB2/3-Ig, for example, which antibody is tested for itsability to act as an agonist of heregulin by binding to a receptorcomplex of two or more ErbB receptors (e.g., an ErbB2/ErbB3 orErbB2/ErbB4 complex) which activates a kinase domain of one or more ofthose receptors and thereby results in phosphorylation of tyrosineresidues in one or more of the receptors, and/or phosphorylation oftyrosine residues in additional substrate polypeptides(s). ErbB receptorphosphorylation can be quantified using the tyrosine phosphorylationassays described below. The phrase “inhibiting an ErbB receptor” refersto the antagonistic property of a chimeric ErbB heteroadhesin or anantagonist antibody raised against it which, when bound to an ErbBreceptor prevents activation of the receptor (ie. inhibits kinasefunction).

The expression “decreasing survival of a cell” refers to the act ofdecreasing the period of existence of a cell, relative to an untreatedcell which has not been exposed to chimeric ErbB-IgG (or an antagonisticantibody raised thereto) either in vitro or in vivo. The expression“decreased cell proliferation” refers to a decrease in the number ofcells in a population exposed to chimeric ErbB-IgG (or an antagonisticantibody raised thereto) either in vitro or in vivo, relative to anuntreated cell.

The expression “increasing survival of a cell” or “increased cellproliferation” refers to increased existence or increased number ofcells in a population exposed to an agonist antibody raised to achimeric ErbB-IgG of the invention, either in vitro or in vivo, relativeto an untreated cell. An increase or decrease in cell proliferation incell culture can be detected by counting the number of cells before andafter exposure to the agonist anti-ErbB-IgG antibody. The extent ofproliferation can be quantified via microscopic examination of thedegree of confluency. Cell proliferation can also be quantified bymeasuring ³H-thymidine uptake by the cells.

By “enhancing differentiation of a cell” is meant the act of increasingthe extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e. cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g. identifying morphologicalchanges in the cell). Enhancing differentiation of a cell also refersherein to cellular maturation in which, for example, unique proteinsassociated with the mature cell are synthesized.

A “glial cell” is derived from the central and peripheral nervous systemand can be selected from oligodendroglial, astrocyte, ependymal, ormicroglial cells as well as satellite cells of ganglia and theneurolemmal cells around peripheral nerve fibers.

“Muscle cells” include skeletal, cardiac or smooth muscle tissue cells.This term encompasses those cells which differentiate to form morespecialized muscle cells (e.g. myoblasts).

“Isolated nucleic acid” is RNA or DNA free from at least onecontaminating source nucleic acid with which it is normally associatedin the natural source and preferably substantially free of any othermammalian RNA or DNA. The phrase “free from at least one contaminatingsource nucleic acid with which it is normally associated” includes thecase where the nucleic acid is present in the source or natural cell butis in a different chromosomal location or is otherwise flanked bynucleic acid sequences not normally found in the source cell. Isolatednucleic acid is RNA or DNA that encodes a biologically active chimericheteromultimer adhesin in which each extracellular domain shares atleast 75%, more preferably at least 80%, still more preferably at least85%, even more preferably 90%, and most preferably 95% sequence identitywith the extracellular domain of the monomer of the natural receptorfrom which it was derived.

“Stringent conditions” are those that (a) employ low ionic strength andhigh temperature for washing, for example, 0.015 M NaCl/0.0015 M sodiumcitrate/0.1% NaDodSO₄ (SDS) at 50° C., or (b) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.

“Moderately stringent conditions” are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and include the use of a washing solution andhybridization conditions (e.g., temperature, ionic strength, and % SDS)less stringent than described above. An example of moderately stringentconditions is a condition such as overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiurncitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc., as necessary to accommodate factors such as probe length and thelike. The expression “control sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, and a ribosome binding site. Eukaryotic cells are known toutilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

An HRG “antagonist” is a molecule which prevents, or interferes with, anHRG effector function (e.g. a molecule which prevents or interferes withbinding and/or activation of an ErbB receptor by HRG). Such moleculescan be screened for their ability to competitively inhibit ErbB receptoractivation by HRG in the tyrosine phosphorylation assay disclosedherein, for example. Preferred antagonists are those which do notsubstantially interfere with the interaction of other heregulinpolypeptides with ErbB receptor(s). Examples of HRG antagonists includeneutralizing antibodies against ErbB2/3-Ig or ErbB2/4-Ig chimericheteroadhesins of the invention.

The term “antibody” is used in the broadest sense and specificallycovers single anti-chimeric heteroadhesin (such as anti-ErbB2/3-IgG oranti-ErbB2/4-IgG) monoclonal antibodies and anti-chimeric heteroadhesinantibody compositions with polyepitopic specificity (includingneutralizing and non-neutralizing antibodies). The antibody ofparticular interest herein is one which does not significantlycross-react with other heteromultimer receptors, such as those describedin the background section above and thus is one which “bindsspecifically” to a heteromultimer receptor, such as ErbB2/3 or ErbB2/4.In such embodiments, the extent of binding of the antibody to non-ErbBreceptors will be less than 10% as determined byradioimmunoprecipitation (RIA), for example.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, ie.,the individual antibodies comprising the population are identical exceptfor possible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-chimeric heteroadhesin antibody with a constant domain(e.g. “humanized” antibodies), or a light chain with a heavy chain, or achain from one species with a chain from another species, or fusionswith heterologous proteins, regardless of species of origin orimmunoglobulin class or subclass designation, as well as antibodyfragments (e.g., Fab, F(ab)₂, and Fv), so long as they exhibit thedesired biological activity. (See, e.g., U.S. Pat. No. 4,816,567 andMage & Lamoyi, in Monoclonal Antibody Production Techniques andApplications, pp. 79-97 (Marcel Dekker, Inc.), New York (1987)).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methods (U.S.Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., Nature 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

By “neutralizing antibody” is meant an antibody molecule as hereindefined which is able to block or significantly reduce an effectorfunction of native sequence HRG. For example, a neutralizing antibodymay inhibit or reduce the ability of HRG to activate an ErbB receptor inthe tyrosine phosphorylation assay described herein. The neutralizingantibody may also block the mitogenic activity of HRG in the cellproliferation assay disclosed herein.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as sheep, dogs, horses, cats, cows, etc. Preferably,the mammal herein is human.

“Pharmaceutically acceptable” carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween™, polyethylene glycol (PEG), and Pluronics™.

II. Modes for Practicing the Invention

1. Production of a Chimeric Heteromultimer Adhesin.

A chimeric heteroadhesin of the invention is preferably produced byexpression in a host cell and isolated therefrom. A host cell isgenerally transformed with the nucleic acid of the invention. Preferablythe nucleic acid is incorporated into an expression vector. Suitablehost cells for cloning or expressing the vectors herein are prokaryotehost cells (such as E. coli, strains of Bacillus, Pseudomonas and otherbacteria), yeast and other eukaryotic microbes, and higher eukaryotecells (such as Chinese hamster ovary (CHO) cells and other mammaliancells). The cells may also be present in live animals (for example, incows, goats or sheep). Insect cells may also be used. Cloning andexpression methodologies are well known in the art.

To obtain expression of a chimeric heteromultimer such as ErbB2-IgG,ErbB3-IgG, and/or ErbB4-IgG, an expression vector is introduced intohost cells by transformation or transfection and the resultingrecombinant host cells are cultured in conventional nutrient media,modified as appropriate for inducing promoters, selecting recombinantcells, or amplifying ErbB-IgG DNA. In general, principles, protocols,and practical techniques for maximizing the productivity of in vitromammalian cell cultures can be found in Mammalian Cell Biotechnology: aPractical Approach, M. Butler, ed. (IRL Press, 1991).

The terms “transformation” and “transfection” are used interchangeablyherein and refer to the process of introducing DNA into a cell.Following transformation or transfection, the nucleic acid of theinvention may integrate into the host cell genome, or may exist as anextrachromosomal element. If prokaryotic cells or cells that containsubstantial cell wall constructions are used as hosts, the preferredmethods of transfection of the cells with DNA is the calcium treatmentmethod described by Cohen, S. N. et al., Proc. Nad. Acad. Sci. U.S.A.,69:2110-2114 (1972) or the polyethylene glycol method of Chung et al.,Nuc. Acids. Res. 16:3580 (1988). If yeast are used as the host,transfection is generally accomplished using polyethylene glycol, astaught by Hinnen, Proc. Natl. Acad. Sci. U.S.A., 75:1929-1933 (1978). Ifmammalian cells are used as host cells, transfection generally iscarried out by the calcium phosphate precipitation method, Graham etal., Virology 52:546 (1978), Gorman et al., DNA and Protein Eng. Tech.2:3-10 (1990). However, other known methods for introducing DNA intoprokaryotic and eukaryotic cells, such as nuclear injection,electroporation, or protoplast fusion also are suitable for use in thisinvention.

Particularly useful in this invention are expression vectors thatprovide for the transient expression in mammalian cells of DNA encodinga chimeric heteroadhesin such as ErbB2/3-Ig or ErbB2/4-Ig. In general,transient expression involves the use of an expression vector that isable to efficiently replicate in a host cell, such that the host cellaccumulates many copies of the expression vector and, in turn,synthesizes high levels of a desired polypeptide encoded by theexpression vector. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties.

A chimeric heteroadhesin preferably is recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates. As a first step, the particulate debris, either host cellsor lysed fragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the chimeric heteroadhesin from other impurities by one ormore purification procedures selected from: fractionation on animmunoaffinity column; fractionation on an ion-exchange column; ammoniumsulphate or ethanol precipitation; reverse phase HPLC; chromatography onsilica; chromatography on heparin Sepharose; chromatography on a cationexchange resin; chromatofocusing; SDS-PAGE; and gel filtration.

Preparation of epitope tagged chimeric heteromultimer, such as ErbB-IgG,facilitates purification using an immunoaffinity column containingantibody to the epitope to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-ErbB column can be employed toabsorb the ErbB4gG by binding it to an ErbB immune epitope.

Amino acid sequence variants of native sequence extracellular domainincluded in the chimeric heteroadhesin are prepared by introducingappropriate nucleotide changes into the native extracellular domain DNAsequence, or by in vitro synthesis of the desired chimeric heteroadhesinmonomer polypeptide. Such variants include, for example, deletions from,or insertions or substitutions of, residues in the amino acid sequenceof the chimeric heteroadhesin.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Seeespecially Table I therein and the discussion surrounding this table forguidance on selecting amino acids to change, add, or delete.

Nucleic acid molecules encoding amino acid sequence variants of nativesequence extracellular domains (such as from ErbB) are prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of native sequence ErbB2, -3, and/or -4.

A preferred type of chimeric amino acid sequence is a fusion proteincomprising an extracellular domain, such as from an ErbB monomer, linkedto a heterologous polypeptide, such as a multimerization domain(immunoglobulin constant region and the like). Such a sequence can beconstructed using recombinant DNA techniques. Alternatively, theheterologous polypeptide can be covalently bound to the extracellulardomain polypeptide by techniques well known in the art such as the useof the heterobifunctional crosslinking reagents. Exemplary couplingagents include N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

In one embodiment, a chimeric heteroadhesin polypeptide comprises afusion of a monomer of the chimeric heteroadhesin with a tag polypeptidewhich provides an epitope to which an anti-tag antibody can selectivelybind. Such epitope tagged forms of the chimeric heteroadhesin areuseful, as the presence thereof can be detected using a labeled antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe chimeric heteroadhesin to be readily purified by affinitypurification using the anti-tag antibody. Tag polypeptides and theirrespective antibodies are well known in the art. Examples include theflu HA tag polypeptide and its antibody 12CA5, (Field et al., Mol. Cell.Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology5(12):3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D(gD) tag and its antibody (Paborsky et al., Protein Engineering3(6):547-553 (1990)).

When preparing the chimeric heteroadhesins of the present invention,nucleic acid encoding an extracellular domain of a naturalheteromultimeric receptor is fused C-terminally to nucleic acid encodingthe N-terminus of an immunoglobulin constant domain sequence, howeverN-terminal fusions are also possible. Typically, in such fusions theencoded chimeric polypeptide will retain at least functionally activehinge, CH2 and CH3 domains of the constant region of an immunoglobulinheavy chain. Fusions are also made to the C-terminus of the Fc portionof a constant domain, or immediately N-terminal to the CH1 of the heavychain or the corresponding region of the light chain. The resultant DNAfusion construct is expressed in appropriate host cells.

Another type of covalent modification of a chimeric heteromultimercomprises linking a monomer polypeptide of the heteromultimer to one ofa variety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. A chimeric heteromultimer also may beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

Generally, the ErbB chimeric heteromultimers of the invention will haveany one or more of the following properties: (a) the ability to competewith a natural heteromultimeric receptor for binding to a neuregulin,such as heregulin; (b) the ability to form ErbB2-IgG/ErbB3-IgG and/orErbB2-IgG/ErbB4-IgG complexes; and (c) the ability to inhibit activationof a natural heteromultimeric receptor by depleting heregulin from theenvironment of the natural receptor, thereby inhibiting proliferation ofcells that express the ErbB2 and ErbB3 receptor and/or the ErbB2 andErbB4 receptor.

To screen for property (a), the ability of the chimeric ErbBheteromultimer adhesin to bind to γ-heregulin can be readily determinedin vitro. For example, immunoadhesin forms of these receptors can begenerated (see below) and the ErbB2/3-Ig or ErbB2/4-Igheteroimmunoadhesin can be immobilized on a solid phase (e.g. on assayplates coated with goat-anti-human antibody). The ability of HRG to bindto the immobilized immunoadhesin can then be determined, e.g. bydetermining competitive displacement by other heregulin molecules. Formore details, see the ¹²⁵I-HRG binding assay described in the Examplebelow.

As to property (c), the tyrosine phosphorylation assay using MCF7 cellsdescribed in the Example provides a means for screening for activationof ErbB receptors. In an alternative embodiment of the invention, theKIRA-ELISA described in WO 95/14930 can be used to qualitatively andquantitatively measure the ability of an ErbB chimeric heteroadhesin toinhibit activation of an ErbB receptor.

The ability of a chimeric heteroadhesin such as ErbB2/3-Ig or ErbB2/4-Igto stimulate proliferation of a cell which expresses the ErbB2 and ErbB3receptor and/or ErbB2 and ErbB4 receptor can readily be determined incell culture. Useful cells for this experiment include MCF7 and SK-BR-3cells obtainable from the ATCC and Schwann cells (see, for example, Liet al., J. Neuroscience 16(6):2012-2019 (1996)). These tumor cell linesmay be plated in cell culture plates and allowed to adhere thereto. TheHRG ligand in the presence and absence of an ErbB chimeric heteroadhesinis added. Monolayers may be washed and stained/fixed with crystalviolet. Cell proliferation or growth inhibition can therefore bequantified as described.

Other heteromultimeric receptors to which the present invention may beapplied for the preparation of useful chimeric heteroadhesins includethe following: Axl, Rse, epidermal grouth factor (EGF) receptor,hepatocyte growht factor (HGF) receptor, IL-2, c-mer, Al-1, EPH, TrkA,TrkB, TrkC, TNF, IL-10, CRF2-4, RXR, RON, AChRα/δ, TRα/RXRα, Trα/DR4,Trα/MHC-TRE, Trα/ME, Trα/F2, KDR/FLT-1, FLT/VEGF, VEGF121/165, Arnt/Ahr,CGA/CGB, EGFR/p185-neu, prolactin receptor (PRL), T cell receptor (TCR),fibroblast growth factor (FGF), Cak receptor, IL-6/gp130, IL-11/gp130leukemia inhibitory factor (LIF)/gp130, cardiotrophin-1/gp130 (CT-1),IL-11/gp130, ciliary neurotrophic factorCNTF/gp130, oncostatin M(OSM)/gp130, interferon γ, and interferon α,β.

A chimeric heteroadhesin of the invention comprises the extracellulardomains of a naturally occurring heteromultimeric receptor, wherein anECD (or ligand binding fragment thereof) of a monomer of the receptor isfused to a multimerization domain as described above. The chimericmonomers of the heteroadhesin stably associated via the multimerizationdomains to form the chimeric heteroadhesin. The heteroadhesins of theinvention bind the ligand of the natural receptor from which the ECDsare obtained and are useful as antagonists of the ligand. Suchantagonists are useful in treating disease states resulting from ligandbinding and activation of the natural heteromultimeric receptor.

2. Therapeutic Compositions and Methods

Use of the chimeric heteroadhesins of the invention as therapeuticcompositions is an embodiment of the invention. The uses generallydisclosed herein are provided as guidance for the use of the chimericheteroadhesins in general. The ErbB chimeric heteroadhesins aredisclosed as examples for firther guidance.

HRG promotes the development, maintenance, and/or regeneration ofneurons in vivo, including central (brain and spinal chord), peripheral(sympathetic, parasympathetic, sensory, and enteric neurons), and motorneurons. Accordingly, an HRG agonist such as an anti-ErbB-Ig antibodyagonist may be utilized in methods for the diagnosis and/or treatment ofa variety of “neurologic diseases or disorders” which Affect the nervoussystem of a mammal, such as a human. According to this embodiment of theinvention, the agonist antibody raised to the ErbB chimericheteroadhesin cross-reacts with and activates the ErbB receptor.

Such diseases or disorders may arise in a patient in whom the nervoussystem has been damaged by, e.g., trauma, surgery, stroke, ischemia,infection, metabolic disease, nutritional deficiency, malignancy, ortoxic agents. The agent is designed to promote the survival,proliferation or differentiation of neurons. For example, anti-ErbBchimeric heteroadhesin agonist antibody can be used to promote thesurvival or proliferation of motor neurons that are damaged by trauma orsurgery. It can also be used to treat motoneuron disorders, such asamyotrophic lateral sclerosis (Lou Gehrig's disease), Bell's palsy, andvarious conditions involving spinal muscular atrophy, or paralysis. Theagonist antibody can be used to treat human “neurodegenerativedisorders”, such as Alzheimer's disease, Parkinson's disease, epilepsy,multiple sclerosis, Huntington's chorea, Down's Syndrome, nervedeafness, and Meniere's disease.

Further, an anti-ErbB chimeric heteroadhesin agonist antibody can beused to treat neuropathy, and especially peripheral neuropathy.“Peripheral neuropathy” refers to a disorder affecting the peripheralnervous system, most often manifested as one or a combination of motor,sensory, sensorimotor, or autonomic neural dysfunction. The wide varietyof morphologies exhibited by peripheral neuropathies can each beattributed uniquely to an equally wide number of causes. For example,peripheral neuropathies can be genetically acquired, can result from asystemic disease, or can be induced by a toxic agent. Examples includebut are not limited to distal sensorimotor neuropathy, or autonomicneuropathies such as reduced motility of the gastrointestinal tract oratony of the urinary bladder. Examples of neuropathies associated withsystemic disease include post-polio syndrome; examples of hereditaryneuropathies include Charcot-Marie-Tooth disease, Refsum's disease,Abetalipoproteinemia, Tangier disease, Krabbe's disease, Metachromaticleukodystrophy, Fabry's disease, and Dejerine-Sottas syndrome; andexamples of neuropathies caused by a toxic agent include those caused bytreatment with a chemotherapeutic agent.

An anti-ErbB chimeric heteroadhesin agonist antibody of the inventionmay also be used to treat muscle cells and medical conditions affectingthem. For example, the HRG may be used to treat a pathophysiologicalcondition of the musculature in a mammal, such as a skeletal muscledisease (e.g. myopathy or dystrophy), a cardiac muscle disorder (such asatrial cardiac arrhythmias, cardiomyopathy, ischemic damage, congenitaldisease, or cardiac trauma), or a smooth muscle disorder (for example,arterial sclerosis, vascular lesion, or congenital vascular disease); totreat muscle damage; to decrease atrophy of muscle cells; to increasemuscle cell survival, proliferation and/or regeneration in a mammal; totreat hypertension; and/or to treat a muscle cell which has insufficientfunctional acetylcholine receptors (as in a patient with myastheniagravis or tachycardia).

An anti-ErbB chimeric heteroadhesin agonist antibody may be used toinduce the formation of ion channels in a surface membrane of a celland/or for enhancing the formation of synaptic junctions in anindividual. HRG may be also useful as a memory enhancer and mayeliminate the “craving” for nicotine.

The anti-ErbB chimeric heteroadheshi agonist antibody may be used toenhance repair and/or regenerate tissues that produce ErbB receptor(s),especially the ErbB2 receptor. For example, the anti-ErbB chimericheteroadhesin agonist antibody may be used to treat dermal wounds;gastrointestinal disease; Barrett's esophagus; cystic or non-cystic endstage kidney disease; and inflammatory bowel disease. Similarly, thismolecule may be used to promote reepithelialization in the humangastrointestinal, respiratory, reproductive or urinary tract.

It may be desirable to treat the mammal with a HRG antagonist, such asan ErbB-Ig chimeric heteroadhesin, particularly where excessive levelsof HRG are present and/or excessive activation of ErbB receptors by HRGis occurring in the mammal. Exemplary conditions or disorders to betreated with a HRG antagonist include benign or malignant tumors (e.g.renal, liver, kidney, bladder, breast, gastric, ovarian, colorectal,prostate, pancreatic, ling, vulval, thyroid, hepatic carcinomas;sarcomas; glioblastomas; and various head and neck tumors); leukemiasand lymphoid malignancies; other disorders such as neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; inflammatory, angiogenic andimmunologic disorders; psoriasis and scar tissue formation. HRGantagonists may also be used to reverse resistance of tumor cells to theimmune-response, to inhibit pathological angiogenesis and to stimulatethe immune system.

In still further embodiments of the invention, an anti-ErbB chimericheteroadhesin as a HRG antagonist may be administered to patientssuffering from neurologic diseases or disorders characterized byexcessive production of HRG and/or excessive ErbB receptor activation byHRG. An anti-ErbB chimeric heteroadhesin antagonist antibody may be usedin the prevention of aberrant regeneration of sensory neurons such asmay occur post-operatively, or in the selective ablation of sensoryneurons, for example, in the treatment of chronic pain syndromes.

There are two major approaches to introducing the nucleic acid(optionally contained in a vector) into the patient's cells; in vivo andex vivo. For in vivo delivery the nucleic acid is injected directly intothe patient, usually at the site where the chimeric heteroadhesin isrequired. For ex vivo treatment, the patient's cells are removed, thenucleic acid is introduced into these isolated cells and the modifiedcells are administered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187).

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

Therapeutic formulations of a chimeric heteroadhesin or an antibodyraised against it are prepared for storage by mixing the heteroadhesinor antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th Edition, Osol., A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Pharmaceutically acceptable carriers, excipients, or stabilizers arenon-toxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, Pluronics™, or polyethylene glycol (PEG).

A chimeric heteroadhesin or anti-chimeric heteroadhesin antibody to beused for in vivo administration must be sterile. This is readilyaccomplished by filtration through sterile filtration membranes, priorto or following lyophilization and reconstitution. The formulationordinarily will be stored in lyophilized form or in solution.

Therapeutic chimeric heteroadhesin or anti-chimeric heteroadhesinantibody compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of chimeric heteroadhesin or antibody administration is inaccord with known methods, e.g., injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes, or by sustained-release systemsas noted below. The heteroadhesin or antibody is administeredcontinuously by infusion or by bolus injection.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981)and Langer, Chem. Tech., 12:98-105 (1982) or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22:547-556 (1983)), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

Sustained-release chimeric heteroadhesin or agonist or antagonistanti-heteroadhesin antibody compositions also include liposomallyentrapped drug. Liposomes containing HRG are prepared by methods knownper se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. % cholesterol, the selected proportion beingadjusted for the optimal therapy. Particularly useful liposomes can begenerated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. A chemotherapeutic agent (such as Doxorubicin) isoptionally contained within the liposome. See Gabizon et al. J. NationalCancer Inst. 81(19):1484 (1989).

The ErbB-Ig chimeric heteroadhesin of the invention may be used to bindand sequester HRG ligand thereby inhibiting ErbB activatin in the celland inhibit a cell proliferation disorder in a patient such as cancer.

A cancer patient to be treated with an ErbB2/3-Ig or ErbB2/4-Igheregulin antagonist or anti-ErbB-Ig antibody as an antagonist asdisclosed herein may also receive radiation therapy. Alternatively, orin addition, a chemotherapeutic agent may be administered to thepatient. Preparation and dosing schedules for such chemotherapeuticagents may be used according to manufacturers' instructions or asdetermined empirically by the skilled practitioner. Preparation anddosing schedules for such chemotherapy are also described inChemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore,Md. (1992). The chemotherapeutic agent may precede, or followadministration of the antagonist or may be given simultaneouslytherewith. For cancer indications, it may be desirable to alsoadminister antibodies against tumor associated antigens or againstantiogenic factors, such as antibodies which bind to EGFR, ErbB2, ErbB3,ErbB4, or vascular endothelial factor (VEGF). Alternatively, or inaddition, one or more cytokines may be co-administered to the patient.

An effective amount of antagonist to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the maximum therapeutic effect.A typical dosage might range from about 1 μg/kg to up to 100 mg/kg ofpatient body weight, preferably about 10 μg/kg to 10 mg/kg. Typically,the clinician will administer antagonist until a dosage is reached thatachieves the desired effect for treatment of the above mentioneddisorders.

3. Non-Therapeutic Methods

An HRG agonist anti-ErbB2/3-Ig antibody or anti-erbB2/4-Ig antibody canbe used for growing cells (such as glial and muscle cells) ex vivo. Itis desirable to have such populations of cells in cell culture forisolation of cell-specific factors e.g. p75^(NGFR) which is a Schwanncell specific marker. Such factors are useful as diagnostic tools or, inthe case of p75^(NGFR), can be used an antigens to generate antibodiesfor diagnostic use. It is also beneficial to have populations ofmammalian cells (e.g. Schwann cells) for use as cellular prostheses fortransplantation into mammalian patients (e.g. into areas of damagedspinal cord in an effort to influence regeneration of interruptedcentral axons, for assisting in the repair of peripheral nerve injuriesand as alternatives to multiple autografts).

In accordance with the in vitro methods of the invention, cellscomprising an ErbB receptor are provided and placed in a cell culturemedium. Suitable tissue culture media are well known to persons skilledin the art and include, but are not limited to, Minimal Essential Medium(MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). Thesetissue culture medias are commercially available from Sigma ChemicalCompany (St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells arethen cultured in the cell culture medium under conditions sufficient forthe cells to remain viable and grow in the presence of an effectiveamount of agonistic antibody. The cells can be cultured in a variety ofways, including culturing in a clot, agar, or liquid culture.

Anti-ErbB-Ig antibodies can be used in the diagnosis of cancerscharacterized by erbB (e.g. erbB2) overexpression and/or amplification,wherein anti-chimeric heteroadhesin antibodies that cross-react with theErbB receptor are used. Such diagnostic assay(s) can be used incombination with other diagnostic/prognostic evaluations such asdetermining lymph node status, primary tumor size, histologic grade,estrogen or progesterone status, tumor DNA content (ploidy), or cellproliferation (S-phrase fraction). See Muss et al., New Eng. J. Med.,330(18):1260-1266 (1994).

The sample as herein defined is obtained, e.g. tissue sample from theprimary lesion of a patient. Formalin-fixed, parffin-embedded blocks areprepared. See Muss et al., supra and Press et al., Cancer Research54:2771-2777 (1994). Tissue sections (e.g. 4 μM) are prepared accordingto known techniques. The extent of anti-ErbB2/3-Ig or anti-erbB2/4-Igantibody binding to the tissue sections is then quantified.

Generally, the chimeric heteroadhesin or the anti-chimeric heteroadhesinantibody will be labeled either directly or indirectly with a detectablelabel. Nunerous labels are available which can be generally grouped intothe following categories:

(a) Radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The γ-HRG orantibody can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, Ed. Coligen et al., WileyPublishers, Vols 1 & 2, for example, and radioactivity can be measuredusing scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the chimeric heteroadhesin orantibody using the techniques disclosed in Current Protocols inImmunology, supra, for example. Fluorescence can be quantified using afluorimeter (Dynatech).

(c) Various enzyme-substrate labels are available and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme maycatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using a DynatechML3000 chemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to proteins are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example: (a)Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate,wherein the hydrogen peroxidase oxidizes a dye precursor (e.g.orthophenylene diamine (OPD) or 3,3′,5,5′-tetramethyl benzidinehydrochloride (TMB)); (b) alkaline phosphatase (AP) withpara-Nitrophenyl phosphate as chromogenic substrate; and (c)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Optionally, the label is indirectly conjugated with the chimericheteroadhesin or anti-CHA antibody. The skilled artisan will be aware ofvarious techniques for achieving this. For example, the CHA or anti-CHAantibody can be conjugated with biotin and any of the three broadcategories of labels mentioned above can be conjugated with avidin, orvice versa. Biotin binds selectively to avidin and thus, the label canbe conjugated with the CHA or anti-CHA antibody in this indirect manner.See, Current Protocols in Immunology, supra, for a review of techniquesinvolving biotin-avidin conjugation. Alternatively, to achieve indirectconjugation of the label with the CHA or anti-CHA antibody, the CHA oranti-CHA antibody is conjugated with a small hapten (e.g. digoxin) andone of the different types of labels mentioned above is conjugated withan anti-hapten antibody (e.g. anti-digoxin antibody). Thus, indirectconjugation of the label with the CHA or anti-CHA antibody can beachieved.

In another embodiment of the invention, the CHA or anti-CHA antibodyneed not be labeled, and the presence thereof can be detected using alabeled anti-CHA or anti-antibody antibody (e.g. conjugated with HRPO).

In the preferred embodiment, the HRG or antibody is labeled with anenzymatic label which catalyzes a color change of a substrate (such astetramethyl benzimidine (TMB), or orthaphenylene diamine (OPD)). Thus,the use of radioactive materials is avoided. A color change of thereagent can be determined spectrophotometrically at a suitablewavelength (e.g. 450 nm for TMB and 490 nm for OPD, with a referencewavelength of 650 nm).

Cells thought capable of expressing a ligand such as HRG are exposed tothe labeled ErbB CHA and the intensity of staining of the cell culturemedium determined. While in vitro analysis is normally contemplated, invivo diagnosis using labeled ErbB CHA conjugated to a detectable moiety(e.g. In for imaging) can also be performed. See, e.g., U.S. Pat. No.4,938,948.

CHAs or anti-CHA antibodies are also useful in a radioimmunoassay,enzyme-linked immunoassay, or radioreceptor assay), in affinitypurification techniques (e.g. for HRG, or for an ErbB receptor such asErbB3 or ErbB4 receptor), and in competitive-type receptor bindingassays when labeled with radioiodine, enzymes, fluorophores, spinlabels, and the like. Thus, CHAs are useful as immunogens for generatinganti-CHA antibodies for diagnostic use.

4. Anti-Chimeric Heteroadhesin Antibodies & Uses thereof

Techniques for generating antibodies, such as polyclonal and monoclonalantibodies are well known in the art. Polyclonal antibodies generallyare raised by immunizing animals with CHA or a fragment thereof(optionally conjugated to a heterologous protein that is immunogenic inthe species to be immunized). Monoclonal antibodies directed toward aCHA may be produced using any method which provides for the productionof antibody molecules by continuous cell lines in culture. Examples ofsuitable methods for preparing monoclonal antibodies include theoriginal hybridoma method of Kohler et al., Nature 256:495497 (1975),and the human B-cell hybridoma method, Kozbor, J., Immunol. 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); andBoerner et al., J. Immunol. 147:86-95 (1991).

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of antibodies). The hybridoma cellsof the invention serve as a preferred source of such DNA. Once isolated,the DNA may be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Morrison, et al., Proc. Natl. Acad.Sci. USA 81:6851 (1984)), or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of an anti-CHAmonoclonal antibody herein.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); and Verhoeyen et al., Science 239:1534-1536 (1988)),by substituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol, 151:2296 (1993); and Chothia and Lesk, J. Mol. Biol. 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA 89:4285 (1992); and Presta et al., J. Immnol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

According to an alternative method for producing human antibodies,transgenic animals (e.g., mice) are available that are capable, uponimmunization, of producing a full repertoire of human antibodies in theabsence of endogenous immunoglobulin production. For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region (J_(H)) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production.Transfer of the human germ-line immunoglobulin gene array in suchgerm-line mutant mice will result in the production of human antibodiesupon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad.Sci. USA, 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993);and Bruggermann et al., Year in Immuno. 7:33 (1993).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimicks someof the properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993).

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for a CHA (preferably the ECDs of the CHA) the otherone is for any other antigen. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies). Procedures described below are useful for the preparationof bispecific antibodies as well as the preparation of multimerizationdomains of the CHAs of the invention.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360). Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents are well known inthe art, and are disclosed in U.S. Pat. No. 4,676,980, along with anumber of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule.Each Fab′ fragment was separately secreted from E. coli and subjected todirected chemical coupling in vitro to form the bispecific antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingerbB and normal human T cells, as well as trigger the lytic activity ofhuman cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al. Proc. Natl. Acad.Sci. USA, 902:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al. J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

To manufacture a neutralizing antibody, antibodies are made using thetechniques for generating these molecules elaborated above. Thepreferred neutralizing antibody is specific for the extracellular domainof the CHA and cross-reacts with the extracellular domain of the naturalheteromultimeric receptor, but does not cross-react with otherreceptors. Following production of a panel of antibodies, the antibodiesare subjected to a screening process in order to identify thosemolecules which meet the desired criteria (i.e. which are able toneutralize a biological activity of the natural heteromultimericreceptor either in vitro or in vivo). For example, the ability of theErbB-Ig CHA to block ErbB activity in any one or more of the assaysdescribed above can be evaluated. Those CHAs or anti-CHA antibodieswhich block the ability of HRG to bind to and/or activate an ErbBreceptor and/or the mitogenic activity of HRG on cells can be selectedas neutralizing CHAs or CHA antibodies.

The antibodies may be coupled to a cytotoxic agent or enzyme (e.g. aprodrug-activating enzyme) in a similar manner to that described abovefor a CHA. Furthermore, the antibodies may be labeled as describedabove, especially where the antibodies are to be used in diagnosticassays.

5. Diagnostic Kits & Articles of Manufacture

Since the invention provides at least two types of diagnostic assay (ie.for detecting cancer using anti-ErbB-Ig antibody, for example, and fordetecting the presence of HRG in a sample using ErbB-Ig, for example) asa matter of convenience, the reagents for these assays can be providedin a kit, i.e., a packaged combination of reagents, for combination withthe sample to be tested. The components of the kit will normally beprovided in predetermined ratios. Thus, a kit may comprise the CHA oranti-CHA antibody labeled directly or indirectly with a suitable label.Where the detectable label is an enzyme, the kit will include substratesand cofactors required by the enzyme (e.g. a substrate precursor whichprovides the detectable chromophore or fluorophore). In addition, otheradditives may be included such as stabilizers, buffers and the like. Therelative amounts of the various reagents may be varied widely to providefor concentrations in solution of the reagents which substantiallyoptimize the sensitivity of the assay. Particularly, the reagents may beprovided as dry powders, usually lyophilized, including excipients whichon dissolution will provide a reagent solution having the appropriateconcentration. The kit also suitably includes instructions for carryingout the bioassay.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agent in the composition is the CHA or an HRGantagonist anti-CHA antibody thereof. The label on, or associated with,the container indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution and dextrose solution.It may further include other materials desirable from a commercial anduser standpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

EXAMPLES

The following examples are offered by way of illustration and not by wayof limitation. The examples are provided so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the compounds, compositions, and methods of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to insure accuracywith respect to numbers used (e.g. amounts, temperature, etc. but someexperimental errors and deviation should be accounted for. Unlessindicated otherwise, parts are parts by weight, temperature is indegrees C., and pressure is at or near atmospheric. The disclosures ofall citations in the specification are expressly incorporated herein byreference.

Example 1 Materials and Methods

This example describes the construction, isolation and biochemicalcharacterization of the ErbB2-IgG, ErbB3-IgG, and ErbB4-IgG chimericamino acid sequences and the resultant chimeric heteromultimers of thepresent invention.

Reagents: The EGF-like domain of HRGβ1₍₁₇₇₋₂₄₄₎ was expressed in E.coli, purified and radioiodinated as described previously (Sliwkowski,M. et al. J. Biol. Chem. 269:14661-14665 (1994)). Full-length rHRGβ1,which was expressed in Chinese hamster ovary cells, was used in Westernblot analysis. The anti-ErbB2 monoclonal antibodies 2C4 and 4D5 havebeen described elsewhere (Fendly et al. Cancer Research 50:1550-1558(1990)).

ErbB2-. ErbB3- and ErbB4-immunoadhesins: A unique Mlu I site wasengineered into a plasmid expressing human IgG heavy chain (pDR, a giftfrom J. Ridgeway and P. Carter, Genentech, Inc.) at the region encodingthe hinge domain of the immunoglobulin. Mlu I sites were also engineeredinto a set of ErbB expression plasmids at the region encoding the ECD/TMjunctions of these receptors. All mutageneses were done using the Kunkelmethod (Kunkel, T., Proc. Natl. Acad. Sci. U.S.A. 82:488 (1985)). TheMlu I sites were utilized to make the appropriate ErbB-IgG fusionconstructs. The fusion junctions of the various ErbB-IgG chimeras were:for ErbB2, E⁶⁴⁶ _(ErbB2)-(TR)-DKTH²²⁴ _(VH); for ErbB3, L⁶³⁶_(ErbB3)-(TR)-DKTH²²⁴ _(VH); for ErbB4, G⁶⁴⁰ _(ErbB4)-(TR)-DKTH²²⁴_(VH), where the amino acid numbering of the ErbB polypeptides isdescribed in Plowman et al. (Plowman, G. D. et al., (1993a) PNAS USA90:1746-1750). The conserved TR sequence is derived from the Mlu I site.The sequence of the Fc region used in the preparation of the fusionconstructs is found in Ellison, J. W. et al. Ellison, J. W. et al.(1982) NAR 10:4071-4079). The final expression constructs were in apRK-type plasmid backbone wherein eukaryotic expression is driven by aCMV promoter (Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)).

To obtain protein for in vitro experiments, adherent HEK-293 cells (ATCCNo. CRL-1573) were transfected with the appropriate expression plasmidsusing standard calcium phosphate methods (Gorman et al., supra and Huanget al., Nucleic Acids Res. 18:937-947 (1990)). Serum-containing mediawas replaced with serum-free media 15 hours post-transfection and thetransfected cells incubated for 5-7 days. The resulting conditionedmedia was harvested and passed through Protein A columns (1 mL PharmaciaHiTrap™). Purified IgG fusions were eluted with 0.1 M citric acid (pH4.2) into tubes containing 1 M Tris pH 9.0. The eluted proteins weresubsequently dialyzed against PBS and concentrated using Centri-prep-30filters (Amicon). Glycerol was added to a final concentration of 25% andthe material stored at −20° C. Concentrations of material weredetermined via a Fc-ELISA. ¹²⁵I-HRG Binding Assay: Binding assays wereperformed in Nunc breakapart immuno-module plates. Plate wells werecoated at 4° C. overnight with 100 μl of 5 μg/ml goat-anti-humanantibody (Boehringer Mannheim) in 50 mM carbonate buffer (pH 9.6).Plates were rinsed twice with 200 μl wash buffer (PBS/0.05% Tween-20™)followed by a brief incubation with 100 μl 1% BSA/PBS for 30 min at roomtemperature. Buffer was removed and each well was incubated with 100 μlIgG fusion protein in 1% BSA/PBS under vigorous side-to-side rotationfor 1 hour. Plates were rinsed three times with wash buffer andcompetitive binding was carried out by adding various amounts of coldcompetitor γ-HRG and ¹²⁵I-HRGβ1 and incubating at room temperature for2-3 hours with vigorous side-to-side rotation. Wells were quickly rinsedthree times with wash buffer, drained and individual wells were countedusing a 100 Series Iso Data γ-counter. Scatchard analysis was performedusing a modified Ligand program (Munson, P. and Robard, D. (1980)Analytical Biochemistry 107:220-239).

³H-Thymidine incorporation assay: Tritiated thymidine incorporationassays were performed in a 96-well format. MCF7-7 cells were plated at10,000 cells/well in 50:50 F12/DMEM (high glucose) 0.1% fetal calf serum(100 mL). Cells were allowed to settle for 3 hours, after which ErbB-IgGfusion proteins and/or heregulin were added to the wells (final volumeof 200 mL) and the plates incubated for 15 hours in a 37° C. tissueculture incubator. Tritiated thymidine was added to the wells (20 mL of1/20 diluted tritiated thymidine stock: Amersham TRA 120 B363, 1 mCi/mL)and the plates incubated a further 3 hours. Tritiated material was thenharvested onto GF/C unifilters (96 well format) using a PackardFiltermate 196 harvester. Filters were counted using a Packard Topcountapparatus.

Example 2 ErbB3-IgG and ErbB4-IgG Proteins Bind HRG

As described above, a series of plasmid constructs were prepared thatpermitted the eukaryotic expression of the extracellular domains (ECDs)of ErbB receptors fused to the constant domains of human IgG. Asdepicted in FIG. 1, these receptor-IgG constructs exist in solution asdisulfide-linked dimers. Homodimeric IgG receptors for ErbB2, ErbB3 andErbB4 were individually expressed in HEK-293 cells and the resultingsecreted receptor fusion proteins were purified by affinitychromatography on protein A. Chen et al. (Chen, X. et al., (1996) J.Biol. Chem. 271:7620-7629) reported a similar construction of thehomodimeric ErbB3- and ErbB4-immunoadhesins, which were used asimmunogens for the generation of receptor-specific monoclonalantibodies. Binding analysis of the chimeric immunoadhesin proteins wasperformed using a microtiter plate format (see Example 1). As shown inFIG. 2, the homodimeric ErbB3-IgG and ErbB4-IgG were capable ofspecifically binding ¹²⁵I-HRG, whereas no discernible binding wasdetected with the ErbB2-IgG construct. Scatchard analysis of HRG bindingto ErbB3-IgG displayed a single affinity binding site with a K_(d) of9.3±2.9 nM. Binding constants for detergent-solubilized ErbB3 expressedin insect cells (Carraway et al., 1994), ErbB3 expressed in COS7 cells(Sliwkowski et al., (1994) supra) and ErbB3 expressed in K562 cellsranged between 0.8 to 1.9 nM. Homodimeric ErbB3-IgG has a higheraffinity constant for HRG than the value of 26 nM recently reported byHoran et al. (Horan et al., (1995) J. Biol. Chem. 270:24604-24608) in ananalysis that used a monovalent soluble ECD of ErbB3. These data suggestthat the optimal conformation of the HRG binding site on ErbB3 may bestabilized by a lipid bilayer. A greater loss in binding affinityrelative to the intact receptor, has also been reported for solubleversions of the EGF receptor (Brown, P. M. et al., (1994) Eur. J.Biochem. 225:223-233; and Zhou, M. et al., (1993) Biochemistry32:8193-8198). The affinity constant measured for the ErbB4-IgG was5.0±0.8 nM. This value is in close agreement with that reported byTzahar et al. of 1.5 nM for full-length ErbB4 expressed in COS7 cells(Tzahar, E. et al., (1994) J. Biol. Chem. 269:25226-25233).

Although neuregulins are a family of proteins arising from alternativeRNA splicing, receptor binding is mediated by the EGF-like motif presentin all active isoforms. Chimeric homodimer immunoadhesins containing theECDs of ErbB3 or ErbB4 bound multiple forms of the neuregulin familyprovided that the EGF-like domain of these proteins were present. Theheregulin variants that bound these homodimers included rHRGβ1₁₋₂₄₄,rHRGβ₁₄₄₋₂₄₄, thioredoxine-HRGβ₁₄₄₋₂₄₄, and tthioredoxine-γHRG.rHRGα₁₋₂₃₉.

Example 3 Heterodimeric ErbB-IgG Fusion Proteins form a High AffinityHRG Binding Site when ErbB2 is Present with ErbB3 or ErbB4

Heterodimeric versions of the receptor-IgG constructs were generated byco-transfecting two expression plasmids encoding different receptorsinto the same cell (see Example 1). The resulting secreted forms of thereceptor-IgGs are mixtures of two types of homodimers and the expectedheterodimer. Three different cotransfections were performed to generatethe following ErbB mixtures: ErbB2/3-IgG, ErbB2/4-IgG and ErbB3/4-IgG.Binding affinities for each of the mixtures were then determined. Asshown in FIG. 3A, a high affinity HRG binding site could be detectedwith the ErbB2-containing heterodimers but not the ErbB3/4-IgG.Scatchard plots of these data were curvilinear for the ErbB2-containingheterodimer mixtures (FIGS. 3B and 3C) suggesting the presence oftwo-distinct types of binding sites (Munson, P. and Robard, D. (1980)Analytical Biochem. 107:220-239). A K_(d) of 0.013 nM was measured forthe high affinity binding site, whereas the low affinity binding sitehad a K_(d) of 12 nM. The high affinity binding constant is in agreementwith the values measured when ErbB3 is expressed in cells containinghigh levels of ErbB2 (Carraway et al., (1994) supra) or when highaffinity HRG binding sites are determined from a 2-site fit of bindingdata in high ErbB3 backgrounds (Sliwkowski et al., (1994) supra).ErbB2/4-IgG (FIG. 3C) also exhibited a similar affinity shift whencompared to the ErbB4-IgG homodimer. The measured affinity constant forthe ErbB2/4-IgG was 0.017 nM. Again using a 2-site fit, a low affinitybinding site K_(d) of 5 nM was measured. This value is in closeagreement with the K_(d) measured for the ErbB4-IgG homodimer. Incontrast, the ErbB3/ErbB4-IgG protein (FIG. 3D) did not display a highaffinity site, but instead a K_(d) of 6 nM was measured, which wascomparable to that seen for the ErbB3-IgG and ErbB4-IgG homodimers. Theformation of a high affinity ligand binding site correlated with theco-expression of the ErbB2 ECD with an ECD of another member of the ErbBfamily, suggesting ErbB2 was required for the formation of a highaffinity site. A summary of binding constants for the ErbB-IgG fusionproteins is shown in Table I. The high affinity binding site that wasformed for the heterodimeric ErbB2/3-IgG or ErbB2/4-IgG protein was300-700 fold higher than for the corresponding homodimeric species.TABLE I Binding Constants For ErbB Homodimer and HeterodimerImmunoadhesins ErbB-IgG Construct Kd (nM) ErbB2 NB* ErbB3 9.24 ± 2.94ErbB4 4.98 ± 0.80 ErbB2/3 0.013 ± 0.004 ErbB2/4 0.017 ± 0.009 ErbB3/45.98 ± 0.70*NB indicates no measurable binding

To further test the hypothesis that ErbB2 was contributing to theformation of the high affinity binding site, the effect of an anti-ErbB2ECD antibody to inhibit high affinity binding to the ErbB immunoadhesinswas examined. Binding reactions were conducted in the presence of anantibody, 2C4, which is specific for the ErbB2 ECD (Lewis, G. D. et al.(1996) Cancer Res. 56:1457-1465; Sliwkowski et al., (1994) supra). Asshown in FIG. 4A, the addition of the 2C4 monoclonal antibody had amarked inhibitory effect on HRG binding for the ErbB2/ErbB3-IgGheterodimer but not for the corresponding ErbB3-IgG homodimer.Similarly, the anti-ErbB2 monoclonal antibody also effected HRG bindingto the ErbB2/ErbB4-IgG heterodimer (FIG. 4B) but not to thecorresponding ErbB4-IgG homodimer. These data indicate that the physicalinteraction of the ECD of ErbB2 with the ECD of either ErbB3 or ErbB4results in the formation of a high affinity growth factor binding sitein this soluble receptor system.

Example 4 ErbB-IgG Fusion Proteins Inhibit the Biological Effects of HRG

Upon HRG treatment, a number of different cell types are known toundergo proliferative responses. The ability of the ErbB-IgG proteins toinhibit HRG-dependent thymidine incorporation was tested in the breastcarcinoma cell line, MCF7 (Lewis et al., (1996) supra). Varyingconcentrations of the different ErbB-IgG proteins were incubated with 1nM rHRG and then added to serum-starved monolayer cultures of MCF7 cells(see Example 1). Following a 24 h incubation, cells were then labeledwith ³H-thymidine to measure DNA synthesis. As shown in FIG. 5, allreceptor fusions capable of HRG binding, inhibited the HRG-mediatedmitogenic response in a dose related manner. The heterodimeric IgGs,ErbB3/2-IgG and ErbB4/2-IgG, were more potent than their correspondinghomodimeric fusion proteins.

Discussion

The Extracellular Domain of ErbB2 Modulates the Binding of HRG to ErbB3and ErbB4

Immunoadhesins offer a number of advantages for in vitro analysis (seeChamow, S. M. and Ashkenazi, A. (1996) Trends in Biotechnology 14:52-60,for review). It is the dimerization capacity of the IgG fusions whichappears to mimic the putative in vivo heterodimerization of the ErbBfamily of receptor resulting in the generation of the high affinityheregulin binding site. HRG binding analysis demonstrated thatheterodimeric mixtures that included ErbB2, i. e., ErbB2/ErbB3-IgG andErbB2/ErbB4-IgG, produced a heregulin binding site with greater than 300fold higher affinity than that seen for ErbB3-IgG or ErbB4-IgGhomodimers or the ErbB3/ErbB4-IgG heterodimer. The low affinity HRGbinding site present in the ErbB3/ErbB4-IgG heterodimer suggests thatthe creation of a high affinity heregulin binding site cannot be made bythe combination of any two different ErbB-IgGs, but rather is specificto ErbB2-IgG containing mixtures. Further evidence for the requirementof ErbB2 to generate this high affinity binding site was determined withmonoclonal antibodies directed against ErbB2 (Lewis et al., (1996)supra; Sliwkowski et al., (1994) supra). When binding studies wereconducted with ErbB2-containing heterodimers in the presence of theseantibodies a significant decrease in HRG binding affinity was observed.

The formation of the HRG-ErbB3-ErbB2 complex occurs sequentially in celllines that express normal levels of these receptors. Specifically, HRGbinds to ErbB3 and ErbB2 is then recruited to this HRG occupiedreceptor. The formation of the complex results in a decrease in thedissociation rate of the ligand, generating a high affinity binding site(Karunagaran, D. et al. (1996) EMBO J. 15:254-264). Now it is reportedthat formation of the high affinity complex also occurred in a solublereceptor system in the absence of transmembrane and intracellulardomains, provided that a dimerization motif was present. In contrast,Horan et al. (Horan, T. et al. (1995) J. Biol. Chem. 270:24604-24608)reported no apparent increase in HRG binding to ErbB3-ECD upon theaddition of ErbB2-ECD. In agreement with those findings, a similarresult is obtained if homodimeric ErbB-IgGs produced from singlytransfected cells were mixed and tested for heregulin binding. Theresultant mixtures of ErbB2-IgG homodimers mixed with homodimers ofErbB3-IgG or ErbB4-IgG did not exhibit any greater ligand affinity thanErbB3-IgG or ErbB4-IgG alone. The dimerization motif supplied by the Fccomponent is thus an important feature in the formation of a highaffinity ligand binding site. Moreover, the flexibility of the hingeregion may also assist in facilitating these receptor-ligandinteractions. Without being limited to any one theory, with intactreceptors embedded in a cell membrane, other motifs, such as thetransmembrane domains or the intracellular domain, may also contributeto the stabilization of ErbB2 containing hetero-oligomeric complexes.

The Role of ErbB2 in an Oligomeric Heregulin-Receptor Signaling Complex.

Ligand-induced receptor oligomerization is a common paradigm forsingle-transmembrane pass receptors (Ullrich, A. and Schlessinger, J.(1990) Cell 61:203-212; and Wells, J. A. (1994) Curr. Opin. Cell Biol.6:163-173). Based on the discovery herein of a soluble chimericheterodimer composed of either ErbB3 or ErbB4 with ErbB2, it isconcluded that such a chimeric heterodimer is sufficient for theformation of a high affinity binding state. Two possible models that areconsistent with these data (FIG. 6) are proposed. The ‘contact’ model isanalogous to that developed for growth hormone and its receptor (Wells,J. A. (1996) PNAS USA 93:1-6 ), except that site 1 resides on ErbB3 orErbB4 and site 2 is contributed by ErbB2. This model predicts that theaffinity for HRG binding to site 1 would be similar to that measured forthe ErbB3 or ErbB4 homodimers. ErbB2 is then recruited to the ErbB3-HRGor ErbB4-HRG complex, and contacts the ErbB3 (or ErbB4)-bound HRG. Theformation of the ErbB3-HRG-ErbB2 complex decreases the dissociation ofHRG and generates the higher affinity binding state. Alternatively, the‘conformation’ model postulates that ErbB2 modulates the interaction ofHRG with ErbB3 or ErbB4, but contact between HRG and ErbB2 does notoccur. In this model the interaction of ErbB2 with ErbB3 or ErbB4 altersthe conformation of these receptors and creates a high affinity bindingstate.

Using chemical cross-linking techniques with radiolabeled HRG on cellsexpressing ErbB3 and ErbB2 (Holmes, W. E. et al., (1992) Science256:1205-1210; Sliwkowski et al., (1994) supra), cross-linked complexescorresponding to proteins with molecular sizes of approximately 190 kDaand greater than 500 kDa were observed. These results suggest that theoligomeric structure of the receptor complex may include multiple copiesof ErbB3 and ErbB2. Moreover, since ErbB3 is devoid of intrinsictyrosine kinase activity (Guy et al., (1994) supra), this hypothesisoffers an explanation for the ligand-dependent increase in tyrosinephosphorylation that is observed for both ErbB2 and ErbB3. For example,a complex that contains two copies of ErbB3 and two copies of ErbB2would allow for phosphorylation of ErbB3, and the transphosphorylationof the secondary ErbB2 receptors as well. TNF receptor homodimerimmunoadhesins (Ashkenazi, A. et al., (1991) PNAS USA 88:10535-10539)appear to mimic the TNF receptor system in which the cell surface TNFreceptor is a trimer (Banner, D. W. et al., (1993) Cell 73-431-445).

Biological Implications of ErbB2 Modulation of ErbB3 and ErbB4

Since ErbB2 was discovered, it has been assumed that a ligand must existwhich solely interacts with and activates ErbB2. Although numerouscandidate proteins have been put forth as putative ligands for ErbB2(reviewed in Hynes, N. E. and Stem, D. F. (1994) Biochem. Biophys. Acta1198:165-184), no protein has been characterized at the molecular levelwhich fulfills this criterion. Other studies have suggested that ErbB2appears to play a multi-faceted role in both EGF and heregulin receptorcomplexes (Earp et al., (1995) supra; Karunagaran et al., (1996) supra.The functions of ErbB2 in these complexes include altering the affinityof the ligand binding domain, contributing a very potent tyrosine kinasecomponent and providing tyrosine residues which upon phosphorylationprovide activation and amplification of various signal transductionpathways. Heregulin activation of ErbB2 is physiologically relevant atneural-muscular junctions (Altiok, N. et al., (1995) EMBO J.14:4258-4266; Chu, G. C. et al., (1995) Neuron 14:329-339; and Jo, S. A.et al., (1995) Nature 373:158-161) and at neural-Schwann cell junctions(Dong, Z. et al. (1995) Neuron 15:585-596; Marchionni, M. A. et al.(1993) Nature 362:312-318; and Morrissey, T. K. et al. (1995) PNAS USA92:1431-1435). In cell culture experiments using human tumor cell lines,several reports have shown that ablating the interaction of ErbB2 witheither ErbB3 or ErbB4 diminishes downstream signaling as well assubsequent biological responses such as growth (Karunagaran et al.,(1996) supra; Lewis et al., (1996) supra; Pinkas-Kramarski, R. et al.,(1996) EMBO J. 15:2452-2467). The concept of ErbB2 as a permanent‘orphan’ receptor (Lonardo et al., 1990) is further supported by recentreports on the phenotypes of the ErbB2 and neuregulin knockouts. In bothcases, mice that are homozygous for either mutation were embryoniclethal near E10.5 (Lee, K.-F. et al. (1995) Nature 378:394-398; andMeyer, D. and Birchmeier, C. (1995) Nature 378:386-390). In each case,the embryos died of a similar cardiac phenotype, the lack of ventriculartrabeculation. Both embryos also had strikingly similar malformations ofthe hindbrain. These observations further suggest that ErbB2 is criticalto transduce HRG signaling. Under normal biological circumstances,ErbB2's sole function appears to be to mediate HRG and EGF ligandresponses as a common member of these receptor complexes.

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

1. A chimeric heteromultimer adhesin comprising: a first amino acidsequence comprising an extracellular domain of a first monomer of anatural heteromultimeric receptor and a multimerization domain; anadditional amino acid sequence comprising an extracellular domain of anadditional monomer of the natural heteromultimeric receptor and amultimerization domain, wherein the extracellular domains of the firstand additional monomers are associated in a cell to form a naturalheteromultimeric receptor which is activated upon binding of a ligand,and wherein the chimeric heteromultimer adhesin has 10⁻¹ to 10⁶ foldaffinity for the ligand relative to a monomer of the receptor or ahomomultimer of the receptor.
 2. The chimeric heteromultimer adhesin ofclaim 1 wherein the chimeric heteromultimer is an antagonist of theligand.
 3. The chimeric heteromultimer adhesin of claim 1 wherein thechimeric heteromultimer adhesin is soluble in an aqueous solution. 4.The chimeric heteromultimer adhesin of claim 1 wherein themultimerization domain of the first amino acid sequence interacts withthe multimerization domain of each additional amino acid sequence toform a heteromultimer.
 5. The chimeric heteromultimer adhesin of claim 1wherein the multimerization domain comprises an immunoglobulin constantregion.
 6. The chimeric heteromultimer adhesin of claim 1 wherein themultimerization domain is selected from the group consisting of aleucine zipper, an amino acid sequence comprising a protuberancecomplementary to an amino acid sequence comprising a hole, a hydrophobicdomain, a hydrophilic domain, and an amino acid sequence comprising afree thiol moiety which reacts to form an intermolecular disulfide bondwith a multimerization domain of an additional amino acid sequence. 7.The chimeric heteromultimer adhesin of claim 1 wherein the first aminoacid sequence comprises an extracellular domain of the ErbB2 receptormonomer or fragment thereof; an additional amino acid sequence comprisesan extracellular domain of the ErbB3 receptor monomer or fragmentthereof; the multimerization domain of the first and additional aminoacids each comprises an immunoglobulin constant region or fragmentthereof; the first and additional amino acid sequences interact via themultimerization domains to form a heterodimer; and the ligand is aneuregulin.
 8. (canceled)
 9. An isolated nucleic acid sequence encodingan amino acid sequence of the chimeric heteromultimer adhesin ofclaim
 1. 10. The isolated nucleic acid sequence of claim 9, wherein theextracellular domain or fragment thereof is from the ErbB2 receptor, andwherein the multimerization domain comprises an immunoglobulin constantregion or fragment thereof.
 11. The isolated nucleic acid sequence ofclaim 9, wherein the extracellular domain or fragment thereof is fromthe ErbB3 receptor, and wherein the multimerization domain comprises animmunoglobulin constant region or fragment thereof.
 12. (canceled) 13.The isolated nucleic acid of claim 9 further comprising a promoteroperably linked to the nucleic acid molecule.
 14. A vector comprisingthe isolated nucleic acid of claim
 13. 15. A host cell comprising thenucleic acid of claim
 9. 16. A host cell comprising the nucleic acid ofclaim
 10. 17. A host cell comprising the nucleic acid of claim
 11. 18. Ahost cell comprising the nucleic acid of claim
 12. 19. A host cellcomprising: a nucleic acid sequence encoding a first amino acid sequenceof the chimeric heteromultimer adhesin of claim 1, and a nucleic acidsequence encoding an additional amino acid sequence of the chimericheteromultimer adhesin of claim
 1. 20. A host cell comprising: a firstisolated nucleic acid sequence encoding the first amino acid sequence ofthe chimeric heteromultimer adhesin of claim 1, wherein theextracellular domain or fragment thereof is from the ErbB2 receptor andwherein the multimerization domain comprises an immunoglobulin constantregion or fragment thereof; and a second isolated nucleic acid sequenceencoding an additional amino acid sequence of the soluble chimericheteromultimer adhesin of claim 1, wherein the extracellular domain orfragment thereof is from the ErbB3 receptor and wherein themultimerization domain comprises an immunoglobulin constant region orfragment thereof.
 21. (canceled)
 22. An antibody to the chimericheteromultimer adhesin of claim 1, wherein the antibody is an antagonistof the ligand, binds to the natural heteromultimeric receptor andinhibits its activation.
 23. An antibody to the chimeric heteromultimeradhesin of claim 1, wherein the antibody is an agonist of the ligand,binds to the natural heteromultimer receptor and activates it.
 24. Amethod of forming a chimeric heteromultimer adhesin-ligand complex in asample comprising the ligand, said method comprising: contacting thechimeric heteromultimer of claim 1 with the sample under conditions suchthat the ligand binds to the heteromultimer to form a chimericheterodimer adhesin-ligand complex.
 25. The method of claim 24 whereinthe complex inhibits binding of the ligand to the naturalheteromultimer.
 26. The method of claim 25 wherein the sample is atissue.
 27. The method of claim 25 wherein the sample is a fluid. 28.The method of claim 27 wherein the fluid is a body fluid.
 29. The methodof claim 28 wherein the body fluid is selected from the group consistingof serum, blood, urine, and lymph.
 30. The method of claim 28 whereinthe sample is in a mammal.
 31. A method of inhibiting naturalheteromultimer receptor activation, the method comprising: contactingthe chimeric heteromultimer adhesin of claim 1 with a sample comprisinga ligand for the natural heteromultimeric receptor and the receptor; andincubating the chimeric heteromultimer adhesin with the ligand to form acomplex such that activation of the natural heteromultimeric receptor bythe ligand is inhibited.
 32. The method of claim 31, wherein the naturalheteromultimeric receptor is ErbB and the chimeric heteromultimeradhesin comprises the extracellular domains of ErbB2 and ErbB3 orfragments thereof.
 33. The method of claim 31, wherein the naturalheteromultimeric receptor is ErbB and the chimeric heteromultimeradhesin comprises the extracellular domains of ErbB2 and ErbB4 orfragments thereof.
 34. A method of inhibiting natural heteromultimerreceptor activation, the method comprising: contacting the antagonistantibody of claim 22 with the natural heteromultimeric receptor to forman antagonist antibody-heteromultimer receptor complex, whereinactivation of the receptor is inhibited.
 35. A method of activating anatural heteromultimeric receptor, the method comprising: contacting theagonist antibody of claim 23 with the natural heteromultimeric receptorto form an agonist antibody-heteromultimeric receptor complex, whereinthe receptor is activated.
 36. A method for the treatment of a diseasestate comprising administering to a mammal in need thereof atherapeutically effective dose of the chimeric heteromultimer adhesin ofclaim
 1. 37. A method for the treatment of a disease state comprisingadministering to a mammal in need thereof a therapeutically effectivedose of the chimeric heteromultimer adhesin of claim
 7. 38. The methodof claim 37 wherein the disease state is selected from the groupconsisting of inflammatory disorder, cancer, neurofibromatosis,peripheral neuropathologies, and cardiac hypertrophy.
 39. The method ofclaim 36 wherein the extracellular domains of the chimericheteromultimer adhesin are from an extracellular domain or fragmentthereof of a natural heteromultimeric receptor selected from the groupconsisting of Axl, Rse, epidermal grouth factor (EGF) receptor,hepatocyte growth factor (HGF) receptor, IL-2, c-mer, Al-1, EPH, TrkA,TrkB, TrkC, TNF, IL-10, CRF2-4, RXR, RON, AChRα/δ, TRα/RXRα, Trα/DR4,Trα/MHC-TRE, Trα/ME, Trα/F2, KDR/FLT-1, FLT/VEGF, VEGF121/165, Arnt/Ahr,CGA/CGB, EGFR/p185-neu, prolactin receptor (PRL), T cell receptor (TCR),fibroblast growth factor (FGF), Cak receptor, IL-6/gp130, IL-11/gp130leukemia inhibitory factor (LIF)/gp130, cardiotrophin-1/gp130 (CT-1),IL-11/gp130, ciliary neurotrophic factor CNTF/gp130, oncostatin M(OSM)/gp130, interferon γ, and interferon α,β.
 40. The method of claim39 wherein the disease state is selected from the group consisting ofinflammatory disorder, cancer, and cardiac disorder.
 41. Apharmaceutically composition comprising the chimeric heteromultimeradhesin of claim 1 and a pharmaceutically acceptable carrier.
 42. Apharmaceutical composition comprising the antagonist antibody of claim22 and a pharmaceutically acceptable carrier.
 43. A pharmaceuticalcomposition comprising the agonist antibody of claim 23 and apharmaceutically acceptable carrier.
 44. An article of manufacture,comprising: a container; a label on said container; and a compositioncontained within said container; wherein the composition comprises thechimeric heteromultimer adhesin of claim 2, and wherein the compositionis effective for antagonizing binding of the ligand to its naturalheteromultimeric receptor, and the label on the container indicates thatthe composition can be used for antagonizing binding of the ligand tothe natural heteromultimeric receptor.
 45. An article of manufacture,comprising: a container; a label on said container; and a compositioncontained within said container; wherein the composition comprises theanti-chimeric heteromultimer adhesin antibody of claim 22, and whereinthe composition is effective for antagonizing binding of the ligand toits natural heteromultimeric receptor, and the label on the containerindicates that the composition can be used for antagonizing binding ofthe ligand to the natural heteromultimeric receptor.
 46. An article ofmanufacture, comprising: a container; a label on said container; and acomposition contained within said container; wherein the compositioncomprises the anti-chimeric heteromultimer adhesin antibody of claim 23,and wherein the composition is effective for agonizing activation of thenatural heteromultimeric receptor, and the label on the containerindicates that the composition can be used for agonizing activation ofthe natural heteromultimeric receptor.